G-CSF conjugates

ABSTRACT

Polypeptide conjugates with G-CSF activity comprising a polypeptide having at least one introduced lysine residue and at least one removed lysine residue compared to the sequence of human G-CSF, and which are conjugated to 2-6 polyethylene glycol moieties. The conjugates have a low in vitro bioactivity, a long in vivo half-life, a reduced receptor-mediated clearance, and provide a more rapid stimulation of production of white blood cells and neutrophils than non-conjugated recombinant human G-CSF.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 11/004,461,filed Dec. 3, 2004 now abandoned, which is a continuation of U.S.application Ser. No. 10/192,294, filed Jul. 10, 2002, now U.S. Pat. No.6,831,158, which is a continuation-in-part of U.S. application Ser. No.09/904,196 filed Jul. 11, 2001, now U.S. Pat. No. 6,555,660, which is acontinuation-in-part of U.S. application Ser. No. 09/760,008, filed Jan.10, 2001, now U.S. Pat. No. 6,646,110, which claims the benefit ofprovisional applications U.S. Ser. No. 60/176,376, filed Jan. 14, 2000,U.S. Ser. No. 60/189,506, filed Mar. 15, 2000, and U.S. Ser. No.60/215,644, filed Jun. 30, 2000, pursuant 35 U.S.C. § 119(e). Thisapplication also claims priority to Danish Patent Application No. PA2002 00447, filed Mar. 22, 2002, Danish Patent Application No. PA 200200708, filed May 8, 2002, Danish Patent Application No. PA 2000 00094,filed Jan. 10, 2000, Danish Patent Application No. PA 2000 00341, filedMar. 2, 2000, and Danish Patent Application No. PA 2000 00943, filedJun. 16, 2000, pursuant 35 U.S.C. § 119(a). The disclosure of eachapplication listed above is incorporated herein in its entirety for allpurposes.

COPYRIGHT NOTIFICATION

Pursuant to 37 C.F.R. §1.71(e), Applicants note that a portion of thisdisclosure contains material which is subject to copyright protection.The copyright owner has no objection to the facsimile reproduction byanyone of the patent document or the patent disclosure, as it appears inthe Patent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

FIELD OF THE INVENTION

The present invention relates to new polypeptides exhibiting granulocytecolony-stimulating factor (G-CSF) activity, to conjugates between apolypeptide exhibiting G-CSF activity and a non-polypeptide moiety, tomethods for preparing such polypeptides or conjugates and the use ofsuch polypeptides or conjugates in therapy, in particular for thetreatment of neutropenia or leukopenia.

BACKGROUND OF THE INVENTION

The process by which white blood cells grow, divide and differentiate inthe bone marrow is called hematopoiesis (Dexter and Spooncer, Ann. Rev.Cell. Biol., 3:423, 1987). Each of the blood cell types arises frompluripotent stem cells. There are generally three classes of blood cellsproduced in vivo: red blood cells (erythrocytes), platelets and whiteblood cells (leukocytes), the majority of the latter being involved inhost immune defense. Proliferation and differentiation of hematopoieticprecursor cells are regulated by a family of cytokines, includingcolony-stimulating factors (CSF's) such as G-CSF and interleukins (Araiet al., Ann. Rev. Biochem., 59:783-836, 1990). The principal biologicaleffect of G-CSF in vivo is to stimulate the growth and development ofcertain white blood cells known as neutrophilic granulocytes orneutrophils (Welte et al., PNAS-USA 82:1526-1530, 1985, Souza et al.,Science, 232:61-65, 1986). When released into the blood stream,neutrophilic granulocytes function to fight bacterial and otherinfection.

The amino acid sequence of human G-CSF (hG-CSF) was reported by Nagataet al. Nature 319:415-418, 1986. hG-CSF is a monomeric protein thatdimerizes the G-CSF receptor by formation of a 2:2 complex of 2 G-CSFmolecules and 2 receptors (Horan et al. (1996), Biochemistry 35(15):4886-96). Aritomi et al. Nature 401:713-717, 1999 have described theX-ray structure of a complex between hG-CSF and the BN-BC domains of theG-CSF receptor. They identify the following hG-CSF residues as beingpart of the receptor binding interfaces: G4, P5, A6, S7, S8, L9, P10,Q1, S12, L15, K16, E19, Q20, L108, D109, D12, T115, T116, Q119, E122,E123, and L124. Expression of rhG-CSF in Escherichia coli, Saccharomycescerevisiae and mammalian cells has been reported (Souza et al., Science232:61-65, 1986, Nagata et al., Nature 319: 415-418, 1986, Robinson andWittrup, Biotechnol. Prog. 11:171-177, 1985).

Leukopenia (a reduced level of white blood cells) and neutropenia (areduced level of neutrophils) are disorders that result in an increasedsusceptibility to various types of infections. Neutropenia can bechronic, e.g. in patients infected with HIV, or acute, e.g. in cancerpatients undergoing chemotherapy or radiation therapy. For patients withsevere neutropenia, e.g. as a result of chemotherapy, even relativelyminor infections can be serious and even life-threatening. Recombinanthuman G-CSF (rhG-CSF) is generally used for treating various forms ofleukopenia/neutropenia. Thus, commercial preparations of rhG-CSF areavailable under the names filgrastim (Gran® and Neupogen®), lenograstim(Neutrogin® and Granocyte®) and nartograstim (Neu-up®). Gran® andNeupogen® are non-glycosylated and produced in recombinant E. colicells. Neutrogin® and Granocyte® are glycosylated and produced inrecombinant CHO cells and Neu-up® is non-glycosylated with five aminoacids substituted at the N-terminal region of intact rhG-CSF produced inrecombinant E. coli cells.

Various protein-engineered variants of hG-CSF have been reported (e.g.U.S. Pat. Nos. 5,581,476, 5,214,132, 5,362,853,and 4,904,584 andRiedhaar-Olson et al. Biochemistry 35: 9034-9041, 1996). Modification ofhG-CSF and other polypeptides so as to introduce at least one additionalcarbohydrate chain as compared to the native polypeptide has beensuggested (U.S. Pat. No. 5,218,092). It is stated that the amino acidsequence of the polypeptide may be modified by amino acid substitution,amino acid deletion or amino acid insertion so as to effect addition ofan additional carbohydrate chain. In addition, polymer modifications ofnative hG-CSF, including attachment of PEG groups, have been reported(Satake-Ishikawa et al., Cell Structure and Function 17:157-160, 1992,U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,824,784, WO 96/11953, WO95/21629, WO 94/20069).

Bowen et al., Experimental Hematology 27 (1999), 425-432 disclose astudy of the relationship between molecule mass and duration of activityof PEG-conjugated G-CSF mutein. An apparent inverse correlation wassuggested between molecular weight of the PEG moieties conjugated to theprotein and in vitro activity, whereas in vivo activities increased withincreasing molecular weight. It is speculated that a lower affinity ofthe conjugates act to increase the half-life, because receptor-mediatedendocytosis is an important mechanism regulating levels of hematopoieticgrowth factors.

The commercially available rhG-CSF has a short-term pharmacologicaleffect and must therefore be administered once a day for the duration ofthe leukopenic state. A molecule with a longer circulation half-lifewould decrease the number of administrations necessary to alleviate theleukopenia and prevent consequent infections. Another, more significantproblem with currently available rG-CSF products is that patients becomeneutropenic after chemotherapy even after administration of G-CSF. Forthese patients, it is important to be able to reduce the duration anddegree of the neutropenic state as much as possible in order to minimizethe risk of serious infections. A further problem is the occurrence ofdose-dependent bone pain. Since bone pain is experienced by patients asa significant side effect of treatment with rG-CSF, it would bedesirable to provide a rG-CSF product that does not cause bone pain,either by means of a product that inherently does not have this effector that is effective in a sufficiently small dose that no bone pain iscaused. Thus, there is clearly a need for improved recombinantG-CSF-like molecules.

With respect to the half-life, one way to increase the circulationhalf-life of a protein is to ensure that clearance of the protein, inparticular via renal clearance and receptor-mediated clearance, isreduced. This may be achieved by conjugating the protein to a chemicalmoiety which is capable of increasing the apparent size, therebyreducing renal clearance and increasing the in vivo half-life.Furthermore, attachment of a chemical moiety to the protein mayeffectively block proteolytic enzymes from physical contact with theprotein, thus preventing degradation by non-specific proteolysis.Polyethylene glycol (PEG) is one such chemical moiety that has been usedin the preparation of therapeutic protein products. Recently, G-CSFmolecule modified with a single, N-terminally linked 20 kDa PEG group(Neulasta™) was approved for sale in the United States. This PEGylatedG-CSF molecule has been shown to have an increased half-life compared tonon-PEGylated G-CSF and thus may be administered less frequently thancurrent G-CSF products, but it does not reduce the duration ofneutropenia significantly compared to non-PEGylated G-CSF. Thus, thereis still substantial room for improvement of the known G-CSF molecules.

A need therefore still exists for providing novel molecules exhibitingG-CSF activity that are useful in the treatment ofleukopenia/neutropenia, and which have are improved in terms of e.g. anincreased half-life and in particular a reduction in the duration ofneutropenia. The present invention relates to such molecules.

BRIEF DISCLOSURE OF THE INVENTION

The present invention relates to specific conjugates comprising apolypeptide exhibiting G-CSF activity and a non-polypeptide moiety,methods for their preparation and their use in medical treatment and inthe preparation of pharmaceuticals. Accordingly, in a first aspect theinvention relates to various specific conjugates comprising apolypeptide exhibiting G-CSF activity and having an amino acid sequencethat differs from the known amino acid sequence of human G-CSF as shownin SEQ ID NO:1 in at least one specified altered amino acid residuecomprising an attachment group for a non-polypeptide moiety, and havingat least one non-polypeptide moiety attached to an attachment group ofthe polypeptide. These conjugates have a substantially reduced in vitrobioactivity compared to that of non-conjugated hG-CSF, whichsurprisingly has been shown to result in a more rapid neutrophilrecovery. The conjugate of the present invention thus has one or moreimproved properties as compared to commercially available rhG-CSF,including increased stimulation of neutrophils, increased functional invivo half-life, increased serum half-life, reduced side effects, reducedimmunogenicity and/or increased bioavailability. Consequently, medicaltreatment with a conjugate of the invention offers a number ofadvantages over the currently available G-CSF compounds.

In a further aspect the invention relates to polypeptides exhibitingG-CSF activity and which form part of a conjugate of the invention. Thepolypeptides of the invention are contemplated to be useful as such fortherapeutic, diagnostic or other purposes, but find particular interestas intermediate products for the preparation of a conjugate of theinvention.

In a further aspect the invention relates to a polypeptide conjugatecomprising a polypeptide exhibiting G-CSF activity, which comprises anamino acid sequence that differs from the amino acid sequence of hG-CSF(with the amino acid sequence shown in SEQ ID NO:1) in at least oneamino acid residue selected from an introduced or removed amino acidresidue comprising an attachment group for a non-polypeptide moiety, anda sufficient number or type of non-polypeptide moieties to provide theconjugate with an increased half-life and/or a more rapid neutrophilrecovery compared to known recombinant G-CSF products.

In a particular aspect the invention relates to a polypeptide conjugateexhibiting G-CSF activity, comprising a polypeptide having thesubstitutions K16R, K34R, K40R, T105K, and S159K, and optionally asubstitution in position H170, e.g. to R, K or Q, relative to the aminoacid sequence of hG-CSF shown in SEQ ID NO:1, or in a correspondingposition relative to an amino acid sequence having at least 80% sequenceidentity with SEQ ID NO:1, and having 2-6, typically 3-6 polyethyleneglycol moieties with a molecular weight of about 1000-10,000 Da attachedto one or more attachment groups of the polypeptide. Where thesesubstitutions are relative to a sequence with at least about 80%sequence identity with SEQ ID NO:1, the degree of sequence identity istypically at least about 90% or 95%, such as at least about 96%, 97%,98% or 99%.

In still further aspects the invention relates to methods for preparinga conjugate of the invention, including nucleotide sequences encoding apolypeptide of the invention, expression vectors comprising such anucleotide sequence, and host cells comprising such a nucleotidesequence or expression vector.

In final aspects the invention relates to a composition comprising aconjugate or polypeptide of the invention, a method for preparing apharmaceutical composition, use of a conjugate or composition of theinvention as a pharmaceutical, and a method of treating a mammal withsuch composition. In particular, the polypeptide, conjugate orcomposition of the invention may be used to prevent infection in cancerpatients undergoing certain types of radiation therapy, chemotherapy,and bone marrow transplantations, to mobilize progenitor cells forcollection in peripheral blood progenitor cell transplantations, fortreatment of severe chronic or relative leukopenia, irrespective ofcause, and to support treatment of patients with acute myeloid leukemia.Additionally, the polypeptide, conjugate or composition of the inventionmay be used for treatment of AIDS or other immunodeficiency diseases aswell as bacterial infections.

DETAILED DISCLOSURE OF THE INVENTION

Definitions

In the context of the present application and invention the followingdefinitions apply:

The term “conjugate” is intended to indicate a heterogeneous moleculeformed by the covalent attachment of one or more polypeptides, typicallya single polypeptide, to one or more non-polypeptide moieties such aspolymer molecules, lipophilic compounds, carbohydrate moieties ororganic derivatizing agents. The term covalent attachment means that thepolypeptide and the non-polypeptide moiety are either directlycovalently joined to one another, or else are indirectly covalentlyjoined to one another through an intervening moiety or moieties, such asa bridge, spacer, or linkage moiety or moieties. Preferably, theconjugate is soluble at relevant concentrations and conditions, i.e.soluble in physiological fluids such as blood. The term “non-conjugatedpolypeptide” may be used about the polypeptide part of the conjugate.

The term “polypeptide” may be used interchangeably herein with the term“protein”.

The “polymer molecule” is a molecule formed by covalent linkage of twoor more monomers, wherein none of the monomers is an amino acid residue,except where the polymer is human albumin or another abundant plasmaprotein. The term “polymer” may be used interchangeably with the term“polymer molecule”. The term is intended to cover carbohydratemolecules, although, normally, the term is not intended to cover thetype of carbohydrate molecule which is attached to the polypeptide by invivo N- or O-glycosylation (as further described below), since suchmolecule is referred to herein as “an oligosaccharide moiety”. Exceptwhere the number of polymer molecule(s) is expressly indicated everyreference to “a polymer”, “a polymer molecule”, “the polymer” or “thepolymer molecule” contained in a polypeptide of the invention orotherwise used in the present invention shall be a reference to one ormore polymer molecule(s).

The term “attachment group” is intended to indicate an amino acidresidue group of the polypeptide capable of coupling to the relevantnon-polypeptide moiety. For instance, for polymer conjugation, inparticular to PEG, a frequently used attachment group is the ε-aminogroup of lysine or the N-terminal amino group. Other polymer attachmentgroups include a free carboxylic acid group (e.g. that of the C-terminalamino acid residue or of an aspartic acid or glutamic acid residue),suitably activated carbonyl groups, oxidized carbohydrate moieties andmercapto groups. Useful attachment groups and their matching non-peptidemoieties are apparent from the table below.

Conjugation Attachment Examples of non- method/- group Amino acidpeptide moiety Activated PEG Reference —NH₂ N-terminal, Polymer, e.g.PEG, mPEG-SPA Shearwater Corp. Lys, His, Arg with amide or imineTresylated Delgado et al., critical group mPEG reviews in TherapeuticDrug Carrier Systems 9(3, 4): 249-304 (1992) —COOH C-term, Asp, Polymer,e.g. PEG, mPEG-Hz Shearwater Corp. Glu with ester or amide groupOligosaccharide In vitro coupling moiety —SH Cys Polymer, e.g. PEG, PEG-Shearwater Corp. with disulfide, vinylsulphone Delgado et al., criticalmaleimide or vinyl PEG-maleimide reviews in Therapeutic sulfone groupDrug Carrier Systems Oligosaccharide In vitro coupling 9(3, 4): 249-304(1992) moiety —OH Ser, Thr, —OH, Oligosaccharide In vivo O-linked Lysmoiety glycosylation PEG with ester, ether, carbamate, carbonate —CONH₂Asn as part of Oligosaccharide In vivo N- an N- moiety glycosylationglycosylation Polymer, e.g. PEG site Aromatic Phe, Tyr, TrpOligosaccharide In vitro coupling residue moiety —CONH₂ GlnOligosaccharide In vitro coupling Yan and Wold, moiety Biochemistry,1984, Jul 31; 23(16): 3759-65 Aldehyde Oxidized Polymer, e.g. PEG,PEGylation Andresz et al., 1978, Ketone oligosaccharide PEG-hydrazideMakromol. Chem. 179: 301, WO 92/16555, WO 00/23114 Guanidino ArgOligosaccharide In vitro coupling Lundblad and Noyes, moiety ChemicalReagents for Protein Modification, CRC Press Inc., Florida, USAImidazole His Oligosaccharide In vitro coupling As for guanidine ringmoiety

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting anN-glycosylation site (with the sequence N-X′-S/T/C-X″, wherein X′ is anyamino acid residue except proline, X″ any amino acid residue which mayor may not be identical to X′ and which preferably is different fromproline, N is asparagine, and S/T/C is either serine, threonine orcysteine, preferably serine or threonine, and most preferablythreonine). Although the asparagine residue of the N-glycosylation siteis where the oligosaccharide moiety is attached during glycosylation,such attachment cannot be achieved unless the other amino acid residuesof the N-glycosylation site are present. Accordingly, when thenon-peptide moiety is an oligosaccharide moiety and the conjugation isto be achieved by N-glycosylation, the term “amino acid residuecomprising an attachment group for the non-peptide moiety” as used inconnection with alterations of the amino acid sequence of thepolypeptide of interest is to be understood as meaning that one or moreamino acid residues constituting an N-glycosylation site are to bealtered in such a manner that either a functional N-glycosylation siteis introduced into the amino acid sequence or removed from saidsequence.

In the present application, amino acid names and atom names (e.g. CA,CB, NZ, N, O, C, etc.) are used as defined by the Protein DataBank (PDB)(www.pdb.org), which is based on the IUPAC nomenclature (IUPACNomenclature and Symbolism for Amino Acids and Peptides (residue names,atom names etc.), Eur. J. Biochem., 138, 9-37 (1984) together with theircorrections in Eur. J. Biochem., 152, 1 (1985). The term “amino acidresidue” is intended to indicate any naturally or non-naturallyoccurring amino acid residue, in particular an amino acid residuecontained in the group consisting of the 20 naturally occurring aminoacids, i.e. alanine (Ala or A), cysteine (Cys or C), aspartic acid (Aspor D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Glyor G), histidine (His or H), isoleucine (Ile or I), lysine (Lys or K),leucine (Leu or L), methionine (Met or M), asparagine (Asn or N),proline (Pro or P), glutamine (Gln or Q), arginine (Arg or R), serine(Ser or S), threonine (Thr or T), valine (Val or V), tryptophan (Trp orW), and tyrosine (Tyr or Y) residues.

The terminology used for identifying amino acid positions/substitutionsis illustrated as follows: F13 indicates position number 13 occupied bya phenylalanine residue in the reference amino acid sequence. F13Kindicates that the phenylalanine residue of position 13 has beensubstituted with a lysine residue. Unless otherwise indicated, thenumbering of amino acid residues made herein is made relative to theamino acid sequence of hG-CSF shown in SEQ ID NO:1. Alternativesubstitutions are indicated with a “/”, e.g. Q67D/E means an amino acidsequence in which glutamine in position 67 is substituted with eitheraspartic acid or glutamic acid. Multiple substitutions are indicatedwith a “+”, e.g. S53N+G55S/T means an amino acid sequence whichcomprises a substitution of the serine residue in position 53 with anasparagine residue and a substitution of the glycine residue in position55 with a serine or a threonine residue.

The term “nucleotide sequence” is intended to indicate a consecutivestretch of two or more nucleotide molecules. The nucleotide sequence maybe of genomic, cDNA, RNA, semisynthetic or synthetic origin, or anycombination thereof.

The term “polymerase chain reaction” or “PCR” refers to the well-knownmethod for amplification of a desired nucleotide sequence in vitro usinga thermostable DNA polymerase.

“Cell”, “host cell”, “cell line” and “cell culture” are usedinterchangeably herein and all such terms should be understood toinclude progeny resulting from growth or culturing of a cell.“Transformation” and “transfection” are used interchangeably to refer tothe process of introducing DNA into a cell.

“Operably linked” refers to the covalent joining of two or morenucleotide sequences, by means of enzymatic ligation or otherwise, in aconfiguration relative to one another such that the normal function ofthe sequences can be performed. For example, the nucleotide sequenceencoding a presequence or secretory leader is operably linked to anucleotide sequence for a polypeptide if it is expressed as a preproteinthat participates in the secretion of the polypeptide: a promoter orenhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, “operably linked” means that the nucleotidesequences being linked are contiguous and, in the case of a secretoryleader, contiguous and in reading phase. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,then synthetic oligonucleotide adaptors or linkers are used, inconjunction with standard recombinant DNA methods.

The term “introduce” refers to introduction of an amino acid residuecomprising an attachment group for a non-polypeptide moiety, inparticular by substitution of an existing amino acid residue, oralternatively by insertion of an additional amino acid residue. The term“remove” refers to removal of an amino acid residue comprising anattachment group for a non-polypeptide moiety, in particular bysubstitution of the amino acid residue to be removed by another aminoacid residue, or alternatively by deletion (without substitution) of theamino acid residue to be removed.

When substitutions are performed in relation to a parent polypeptide,they are preferably “conservative substitutions”, in other wordssubstitutions performed within groups of amino acids with similarcharacteristics, e.g. small amino acids, acidic amino acids, polar aminoacids, basic amino acids, hydrophobic amino acids and aromatic aminoacids.

Preferred substitutions in the present invention may in particular bechosen from among the conservative substitution groups listed in thetable below.

Conservative Substitution Groups:

1 Alanine (A) Glycine (G) Serine (S) Threonine (T) 2 Aspartic acid (D)Glutamic acid (E) 3 Asparagine (N) Glutamine (Q) 4 Arginine (R)Histidine (H) Lysine (K) 5 Isoleucine (I) Leucine (L) Methionine (M)Valine (V) 6 Phenylalanine (F) Tyrosine (Y) Tryptophan (W)

The term “immunogenicity” as used in connection with a given substanceis intended to indicate the ability of the substance to induce aresponse from the immune system. The immune response may be a cell orantibody mediated response (see, e.g., Roitt: Essential Immunology(8^(th) Edition, Blackwell) for further definition of immunogenicity).Normally, reduced antibody reactivity will be an indication of reducedimmunogenicity. The reduced immunogenicity may be determined by use ofany suitable method known in the art, e.g. in vivo or in vitro.

The term “functional in vivo half-life” is used in its normal meaning,i.e. the time at which 50% of the biological activity of the polypeptideor conjugate is still present in the body/target organ, or the time atwhich the activity of the polypeptide or conjugate is 50% of the initialvalue. As an alternative to determining functional in vivo half-life,“serum half-life” may be determined, i.e. the time in which 50% of thepolypeptide or conjugate molecules circulate in the plasma orbloodstream prior to being cleared. Alternative terms to serum half-lifeinclude “plasma half-life”, “circulating half-life”, “serum clearance”,“plasma clearance” and “clearance half-life”. The polypeptide orconjugate is cleared by the action of one or more of thereticuloendothelial systems (RES), kidney, spleen or liver, byreceptor-mediated degradation, or by specific or non-specificproteolysis, in particular by the action of receptor-mediated clearanceand renal clearance. Normally, clearance depends on size (relative tothe cutoff for glomerular filtration), charge, attached carbohydratechains, and the presence of cellular receptors for the protein. Thefunctionality to be retained is normally selected from proliferative orreceptor-binding activity. The functional in vivo half-life and theserum half-life may be determined by any suitable method known in theart as further discussed in the Materials and Methods section below.

The term “increased” as used about the functional in vivo half-life orserum half-life is used to indicate that the relevant half-life of theconjugate or polypeptide is statistically significantly increasedrelative to that of a reference molecule, such as a non-conjugatedhG-CSF (e.g. Neupogen®) as determined under comparable conditions. Forinstance, the relevant half-life may increased by at least about 25%,such as by at least about 50%, e.g. by at least about 100%, 200%, 500%or 1000%.

The term “renal clearance” is used in its normal meaning to indicate anyclearance taking place by the kidneys, e.g. by glomerular filtration,tubular excretion or tubular elimination. Renal clearance depends onphysical characteristics of the conjugate, including size (diameter),symmetry, shape/rigidity and charge. Reduced renal clearance may beestablished by any suitable assay, e.g. an established in vivo assay.Typically, renal clearance is determined by administering a labeled(e.g. radioactive or fluorescent labeled) polypeptide conjugate to apatient and measuring the label activity in urine collected from thepatient. Reduced renal clearance is determined relative to acorresponding reference polypeptide, e.g. the correspondingnon-conjugated polypeptide, a non-conjugated corresponding wild-typepolypeptide or another conjugated polypeptide (such as a conjugatedpolypeptide not according to the invention), under comparableconditions. Preferably, the renal clearance rate of the conjugate isreduced by at least 50%, preferably by at least 75%, and most preferablyby at least 90% compared to a relevant reference polypeptide.

Generally, activation of the receptor is coupled to receptor-mediatedclearance (RMC) such that binding of a polypeptide to its receptorwithout activation does not lead to RMC, while activation of thereceptor leads to RMC. The clearance is due to internalization of thereceptor-bound polypeptide with subsequent lysosomal degradation.Reduced RMC may be achieved by designing the conjugate so as to be ableto bind and activate a sufficient number of receptors to obtain optimalin vivo biological response and avoid activation of more receptors thanrequired for obtaining such response. This may be reflected in reducedin vitro bioactivity and/or increased off-rate. In a preferredembodiment, the conjugates of the invention have a substantially reducedin vitro bioactivity compared to that of non-conjugated hG-CSF.

Typically, reduced in vitro bioactivity reflects reducedefficacy/efficiency and/or reduced potency and may be determined by anysuitable method for determining any of these properties. For instance,in vitro bioactivity may be determined in a luciferase based assay(“Primary assay 2”; see Materials and Methods). Another method fordetermining the in vitro bioactivity is to determine the bindingaffinity of a conjugate of the invention using the cell-based assaydescribed in the Materials and Methods section (“Secondary assay”).

It has been found that a relatively low in vitro bioactivity, comparedto the activity of hG-CSF (SEQ ID NO:1), is advantageous in terms ofboth a long plasma half-life and a high degree of stimulation ofneutrophils. Surprisingly, it has been found that administration ofG-CSF conjugates of the invention having a low in vitro bioactivityresults in a faster neutrophil recovery, i.e. a faster recovery of theneutrophil count to a normal level, than administration of hG-CSF. Sinceit is critical to be able to reduce the duration of neutropenia as muchas possible in patients having a reduced neutrophil level due to e.g.chemotherapy or radiation therapy, this is an important finding. Thus,in a preferred embodiment, the in vitro bioactivity of a conjugate ofthe invention is in the range of about 2-30%, preferably about 3-25%, ofthe bioactivity of hG-CSF (where the hG-CSF used as the referencepolypeptide has SEQ ID NO:1, optionally with an N-terminal methionineresidue; the reference hG-CSF may in particular be Neupogen®, i.e.non-glycosylated Met-hG-CSF) as determined by the luciferase assaydescribed herein, or, alternatively, using the cell-based receptorbinding affinity assay (“Secondary assay”). The in vitro bioactivity ofthe conjugate is thus preferably reduced by at least 70%, such as by atleast 75%, e.g. by at least 80% or 85%, as compared to the in vitrobioactivity of hG-CSF, determined under comparable conditions. Expresseddifferently, the conjugate may have an in vitro bioactivity that is assmall as about 2%, typically at least about 3%, such as at least about4% or 5%, of that of the wild-type polypeptide. For instance, the invitro bioactivity may be in the range of about 4-20% of that of hG-CSF,determined under comparable conditions. In cases where reduced in vitrobioactivity is desired in order to reduce receptor-mediated clearance,it will be clear that sufficient bioactivity to obtain the desiredreceptor activation must nevertheless be maintained, which is why thebioactivity should be at least about 2% of that of hG-CSF and preferablyslightly higher as explained above.

It has been found that amino acid alterations, in particularsubstitutions, in the helix regions of G-CSF, i.e. in an amino acidresidue selected from amino acid position 11-41 (helix A), 71-95 (helixB), 102-125 (helix C), and 145-170 (helix D) (compared to SEQ ID NO:1),result in a reduced receptor-mediated clearance and thus an increased invivo half-life when the resulting polypeptides are conjugated topolyethylene glycol. In addition to a longer half-life, it hassurprisingly been found that administration of such polypeptideconjugates is able to stimulate production of white blood cells andneutrophils to the same degree as, or even better than, administrationof the commercially available G-CSF products Neupogen® and Neulasta™.G-CSF conjugates having a reduced in vitro bioactivity may thus beprepared by altering, typically by substitution, one or more amino acidresidues in a helix region of G-CSF, and by conjugating the resultingpolypeptide to one or more non-polypeptide moieties such as polyethyleneglycol.

Preferably, the off-rate between the polypeptide conjugate and itsreceptor is increased by a magnitude resulting in the polypeptideconjugate being released from its receptor before any substantialinternalization of the receptor-ligand complex has taken place. Thereceptor-polypeptide binding affinity may be determined as described inthe Materials and Methods section herein. The off-rate may be determinedusing the Biacore® technology as described in the Materials and Methodssection. The in vitro RMC may be determined by labeling (e.g.radioactive or fluorescent labeling) the polypeptide conjugate,stimulating cells comprising the receptor for the polypeptide, washingthe cells, and measuring label activity. Alternatively, the conjugatemay be exposed to cells expressing the relevant receptor. After anappropriate incubation time the supernatant is removed and transferredto a well containing similar cells. The biological response of thesecells to the supernatant is determined relative to a non-conjugatedpolypeptide or another reference polypeptide, and this is a measure ofthe extent of the reduced RMC.

Normally, reduced in vitro bioactivity of the conjugate is obtained as aconsequence of its modification by a non-polypeptide moiety. However, inorder to further reduce in vitro bioactivity or for other reasons it maybe of interest to modify the polypeptide part of the conjugate further.For instance, in one embodiment at least one amino acid residue locatedat or near a receptor binding site of the polypeptide may be substitutedwith another amino acid residue as compared to the correspondingwild-type polypeptide so as to obtain reduced in vitro bioactivity. Theamino acid residue to be introduced by substitution may be any aminoacid residue capable of reducing in vitro bioactivity of the conjugate.Conveniently, the introduced amino acid residue comprises an attachmentgroup for the non-polypeptide moiety as defined herein. In particular,when the non-polypeptide moiety is a polymer molecule such as PEGmolecule, the amino acid residue to be introduced may be a lysineresidue.

The term “exhibiting G-CSF activity” is intended to indicate that thepolypeptide or conjugate has one or more of the functions of nativeG-CSF, in particular hG-CSF with the amino acid sequence shown in SEQ IDNO:1, including the capability to bind to a G-CSF receptor (Fukunaga etal., J. Bio. Chem, 265:14008, 1990). The G-CSF activity is convenientlyassayed using the primary assay described in the Materials and Methodssection hereinafter. The polypeptide “exhibiting” G-CSF activity isconsidered to have such activity when it displays a measurable function,e.g. a measurable proliferative activity or a receptor binding activity(e.g. as determined by the primary assay described in the Materials andMethods section). The polypeptide exhibiting G-CSF activity may also betermed “G-CSF molecule” herein for the sake of simplicity, even thoughsuch polypeptides are in fact variants of G-CSF.

The term “parent G-CSF” or “parent polypeptide” is intended to indicatethe molecule to be modified in accordance with the present invention.The parent G-CSF is normally hG-CSF or a variant thereof. A “variant” isa polypeptide which differs in one or more amino acid residues from aparent polypeptide, normally in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 amino acid residues. Examples of rhG-CSF include filgrastim(Gran® and Neupogen®), lenograstim (Neutrogin® and Granocyte®) andnartograstim (Neu-up®).

Conjugate of the Invention

As stated above, in a first aspect the invention relates to a conjugatecomprising a polypeptide exhibiting G-CSF activity, which comprises anamino acid sequence that differs from the amino acid sequence of SEQ IDNO:1 in at least one amino acid residue selected from specifiedintroduced or removed amino acid residues comprising an attachment groupfor a non-polypeptide moiety, and at least one non-polypeptide moietyattached to an attachment group of the polypeptide. The amino acidresidues to be introduced and/or removed are described in further detailin the following sections. It will be understood that the conjugateitself also exhibits G-CSF activity.

By removing and/or introducing an amino acid residue comprising anattachment group for the non-polypeptide moiety it is possible tospecifically adapt the polypeptide so as to make the molecule moresusceptible to conjugation to the non-polypeptide moiety of choice, tooptimize the conjugation pattern (e.g. to ensure an optimal distributionof non-polypeptide moieties on the surface of the G-CSF molecule and toensure that only the attachment groups intended to be conjugated arepresent in the molecule) and thereby obtain a new conjugate moleculewhich has G-CSF activity and in addition one or more improved propertiesas compared to G-CSF molecules available today.

While the polypeptide may be of any origin, in particular mammalianorigin, it is presently preferred to be of human origin, in particular avariant of a polypeptide having the amino acid sequence of SEQ ID NO:1.

In preferred embodiments of the present invention more than one aminoacid residue of the polypeptide with G-CSF activity is altered, e.g. thealteration embraces removal as well as introduction of amino acidresidues comprising an attachment group for the non-polypeptide moietyof choice.

In addition to the amino acid alterations disclosed herein aimed atremoving and/or introducing attachment sites for the non-polypeptidemoiety, it will be understood that the amino acid sequence of thepolypeptide of the invention may if desired contain other alterationsthat need not be related to introduction or removal of attachment sites,i.e. other substitutions, insertions or deletions. These may, forexample, include truncation of the N- and/or C-terminus by one or moreamino acid residues, or addition of one or more extra residues at the N-and/or C-terminus, e.g. addition of a methionine residue at theN-terminus.

The conjugate of the invention has one or more of the following improvedproperties as compared to hG-CSF, in particular as compared to rhG-CSF(e.g. filgrastim, lenograstim or nartograstim) or known hG-CSF variants:increased ability to reduce the duration of neutropenia, increasedfunctional in vivo half-life, increased serum half-life, reduced renalclearance, reduced receptor-mediated clearance, reduced side effectssuch as bone pain, and reduced immunogenicity.

It will be understood that the amino acid residue comprising anattachment group for a non-polypeptide moiety, whether it be removed orintroduced, will be selected on the basis of the nature of thenon-polypeptide moiety of choice and, in most instances, on the basis ofthe method by which conjugation between the polypeptide and thenon-polypeptide moiety is to be achieved. For instance, when thenon-polypeptide moiety is a polymer molecule such as a polyethyleneglycol or polyalkylene oxide derived molecule amino acid residuescomprising an attachment group may be selected from the group consistingof lysine, cysteine, aspartic acid, glutamic acid, histidine andarginine. When conjugation to a lysine residue is to be achieved, asuitable activated molecule is e.g. mPEG-SPA from Shearwater Corp.,oxycarbonyl-oxy-N-dicarboxyimide-PEG (U.S. Pat. No. 5,122,614), or PEGavailable from PolyMASC Pharmaceuticals plc. The first of these will beillustrated further below.

In order to avoid too much disruption of the structure and function ofthe parent hG-CSF molecule, the total number of amino acid residues tobe altered in accordance with the present invention, e.g. as describedin the subsequent sections herein, (as compared to the amino acidsequence shown in SEQ ID NO:1) will typically not exceed 15. The exactnumber of amino acid residues and the type of amino acid residues to beintroduced or removed depends in particular on the desired nature anddegree of conjugation (e.g. the identity of the non-polypeptide moiety,how many non-polypeptide moieties it is desirable or possible toconjugate to the polypeptide, where conjugation is desired or should beavoided, etc.). Preferably, the polypeptide part of the conjugate of theinvention or the polypeptide of the invention comprises an amino acidsequence which differs in 1-15 amino acid residues from the amino acidsequence shown in SEQ ID NO:1, typically in 2-10 amino acid residues,e.g. in 3-8 amino acid residues, such as 4-6 amino acid residues, fromthe amino acid sequence shown in SEQ ID NO:1. Thus, normally thepolypeptide part of the conjugate or the polypeptide of the inventioncomprises an amino acid sequence which differs from the amino acidsequence shown in SEQ ID NO:1 in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15 amino acid residues.

The polypeptide part of the conjugate will typically have an amino acidsequence with at least about 80% identity with SEQ ID NO:1, preferablyat least about 90%, such as at least about 95%, e.g. at least about 96%,97%, 98% or 99% sequence identity with SEQ ID NO:1. Amino acid sequencehomology/identity is conveniently determined from aligned sequences,using e.g. the ClustalW program, version 1.8, June 1999, using defaultparameters (Thompson et al., 1994, ClustalW: Improving the sensitivityof progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice, Nucleic AcidsResearch, 22: 4673-4680) or from the PFAM families database version 4.0(http://pfam.wustl.edu/) (Nucleic Acids Res. 1999 Jan. 1; 27(1):260-2)by use of GENEDOC version 2.5 (Nicholas, K. B., Nicholas H. B. Jr., andDeerfield, D. W. II. 1997 GeneDoc: Analysis and Visualization of GeneticVariation, EMBNEW.NEWS 4:14; Nicholas, K. B. and Nicholas H. B. Jr. 1997GeneDoc: Analysis and Visualization of Genetic Variation).

In a preferred embodiment one difference between the amino acid sequenceof the polypeptide and the amino acid sequence shown in SEQ ID NO:1 isthat at least one and often more, e.g. 1-15, amino acid residuescomprising an attachment group for the non-polypeptide moiety has beenintroduced, preferably by substitution, into the amino acid sequence.Thereby, the polypeptide part is altered in the content of the specificamino acid residues to which the non-polypeptide moiety of choice binds,whereby a more efficient, specific and/or extensive conjugation isachieved. For instance, when the total number of amino acid residuescomprising an attachment group for the non-polypeptide of choice isaltered to an optimized level, the clearance of the conjugate istypically significantly reduced, due to the altered shape, size and/orcharge of the molecule achieved by the conjugation. Furthermore, whenthe total number of amino acid residues comprising an attachment groupfor the non-polypeptide of choice is increased, a greater proportion ofthe polypeptide molecule is shielded by the non-polypeptide moieties ofchoice, leading to a lower immune response.

The term “one difference” as used in the present application is intendedto allow for additional differences being present. Accordingly, inaddition to the specified amino acid difference, other amino acidresidues than those specified may be mutated.

In a further preferred embodiment one difference between the amino acidsequence of the polypeptide and the amino acid sequence shown in SEQ IDNO:1 is that at least one and preferably more, e.g. 1-15, amino acidresidues comprising an attachment group for the non-polypeptide moietyhas/have been removed, preferably by substitution, from the amino acidsequence. By removing one or more amino acid residues comprising anattachment group for the non-polypeptide moiety of choice it is possibleto avoid conjugation to the non-polypeptide moiety in parts of thepolypeptide in which such conjugation is disadvantageous, e.g. in aminoacid residues located at or near a functional site of the polypeptide(since conjugation at such a site may result in inactivation or reducedG-CSF activity of the resulting conjugate due to impaired receptorrecognition). In the present context the term “functional site” isintended to indicate one or more amino acid residues which is/areessential for or otherwise involved in the function or performance ofhG-CSF. Such amino acid residues are a part of the functional site. Thefunctional site may be determined by methods known in the art and ispreferably identified by analysis of a structure of the polypeptidecomplexed to a relevant receptor, such as the hG-CSF receptor (SeeAritomi et al. Nature 401:713-717, 1999).

In a still further preferred embodiment, the amino acid sequence of thepolypeptide differs from the amino acid sequence shown in SEQ ID NO:1 inthat a) at least one specified amino acid residue comprising anattachment group for the non-polypeptide moiety and present in the aminoacid sequence shown in SEQ ID NO:1 has been removed, preferably bysubstitution, and b) at least one specified amino acid residuecomprising an attachment group for the non-polypeptide moiety has beenintroduced into the amino acid sequence, preferably by substitution, thespecified amino acid residues being any of those described in thesubsequent sections herein. This embodiment is considered of particularinterest in that it is possible to specifically design the polypeptideso as to obtain an optimal conjugation to the non-polypeptide moiety ofchoice. For instance, by introducing and removing selected amino acidresidues as disclosed in the following sections it is possible to ensurean optimal distribution of attachment groups for the non-polypeptidemoiety of choice, which gives rise to a conjugate in which thenon-polypeptide moieties are placed so as to a) effectively shieldepitopes and other surface parts of the polypeptide and b) ensure anoptimal Stokes radius of the conjugate, without causing too muchstructural disruption and thereby impair the function of thepolypeptide.

The conjugate of the invention will in general comprise a sufficientnumber and type of non-polypeptide moieties to provide the conjugatewith an increased functional in vivo half-life and/or serum half-life ascompared to hG-CSF, e.g. filgrastim, lenograstim or nartograstim, andpreferably as compared to rhG-CSF comprising a single N-terminallyattached 20 kDa PEG moiety. The increased functional in vivo half-lifeis conveniently determined as described in the Materials and Methodssection herein.

The conjugate of the invention may comprise at least one non-conjugated,conjugatable attachment group for the non-polypeptide moiety. In thepresent context the term “conjugatable attachment group” is intended toindicate an attachment group that is located in a position of thepolypeptide where it is accessible for conjugation, and that but forspecial precautions is conjugated to the relevant non-polypeptide moietywhen subjected to conjugation. For instance, such attachment group maybe part of an amino acid residue involved in or otherwise essential forthe polypeptide to exert its activity. A convenient way to avoidconjugation of an otherwise conjugatable attachment group is to shieldthe attachment group by means of a helper molecule, e.g. as described inthe section entitled “Blocking of the functional site”. It will beunderstood that the number of non-conjugated, conjugatable attachmentgroups depends on the specific G-SCF polypeptide and the location of theconjugatable attachment groups. For instance, the polypeptide conjugatecomprises one or two non-conjugated, conjugatable attachment groups, andat least one, and preferably two or more conjugated attachment groups.

The four helices of G-CSF comprise amino acid residues 11-41 (helix A),71-95 (helix B), 102-125 (helix C), and 145-170 (helix D) (Zink et al.(1994) Biochemistry 33: 8453-8463). Surprisingly, it has been found thatadvantageous results may be obtained when non-polypeptide moieties areattached to amino acid residues located in one or more of the helices ofG-CSF, even though modification of protein helices, e.g. the helixstructures of four-helix bundle proteins such as G-CSF, is generallyconsidered to be accompanied by a risk of disturbance of proteinfunction. In one embodiment, the polypeptide conjugate of the inventiontherefore comprises at least one non-polypeptide moiety attached to anattachment group of an amino acid residue located in one of the fourhelices, in particular in one or more of the B, C or D helices

Conjugate of the Invention Wherein the Non-Polypeptide Moiety isAttached to a Lysine or the N-Terminal Amino Acid Residue

In one aspect the invention relates to a polypeptide conjugatecomprising

-   i) a polypeptide exhibiting G-CSF activity, comprising an amino acid    sequence that differs from the amino acid sequence shown in SEQ ID    NO:1 in at least one substitution selected from the group consisting    of T1K, P2K, L3K, G4K, P5K, A6K, S7K, S8K, L9K, P10K, Q11K, S12K,    F13K, L14K, L15K, E19K, Q20K, V21K, Q25K, G26K, D27K, A29K, A30K,    E33K, A37K, T38K, Y39K, L41K, H43K, P44K, E45K, E46K, V48K, L49K,    L50K, H52K, S53K, L54K, I56K, P57K, P60K, L61K, S62K, S63K, P65K,    S66K, Q67K, A68K, L69K, Q70K, L71K, A72K, G73K, S76K, Q77K, L78K,    S80K, F83K, Q86K, G87K, Q90K, E93K, G94K, S96K, P97K, E98K, L99K,    G100K, P101K, T102K, D104K, T105K, Q107K, L108K, D109K, A111K,    D112K, F113K, T115K, T116K, W118K, Q119K, Q120K, M121K, E122K,    E123K, L124K, M126K, A127K, P128K, A129K, L130K, Q131K, P132K,    T133K, Q134K, G135K, A136K, M137K, P138K, A139K, A141K, S142K,    A143K, F144K, Q145K, S155K, H156K, Q158K, S159K, L161K, E162K,    V163K, S164K, Y165K, V167K, L168K, H170K, L171K, A172K, Q173K and    P174K, and-   ii) at least one non-polypeptide moiety attached to a lysine residue    of the polypeptide.

hG-CSF contains four lysine residues, of which K16 is located in thereceptor-binding domain and the others are located in positions 23, 34and 40, respectively, all relatively close to the receptor-bindingdomain. In order to avoid conjugation to one or more of these lysineresidues (since this may inactivate or severely reduce the activity ofthe resulting conjugate) it may be desirable to remove at least onelysine residue, e.g. two, three or all of these residues. Accordingly,in another, more preferred aspect the invention relates to a polypeptideconjugate as defined above, wherein at least one of the amino acidresidues selected from the group consisting of K16, K23, K34 and K40 hasbeen deleted or substituted with another amino acid residue. Preferably,at least K16 is substituted with another amino acid residue.

Examples of preferred amino acid substitutions include one or more ofQ70K, Q90K, T105K, Q120K, T133K, S159K and H170K/Q/R, such as two,three, four or five of these substitutions, for example: Q70K+Q90K,Q70K+T105K, Q70K+Q120K, Q70K+T133K, Q70K+S159K, Q70K+H170K, Q90K+T105K,Q90K+Q120K, Q90K+T133K, Q90K+S159K, Q90K+H170K, T105K+Q120K,T105K+T133K, T105K+S159K, T105K+H170K, Q120K+T133K, Q120K+S159K,Q120K+H170K, T133K+S159K, T133K+H170K, S159K+H170K, Q70K+Q90K+T105K,Q70K+Q90K+Q120K, Q70K+Q90K+T133K, Q70K+Q90K+S159K, Q70K+Q90K+H170K,Q70K+T105K+Q120K, Q70K+T105K+T133K, Q70K+T105K+S159K, Q70K+T105K+H170K,Q70K+Q120K+T133K, Q70K+Q120K+S159K, Q70K+Q120K+H170K, Q70K+T133K+S159K,Q70K+T133K+H170K, Q70K+S159K+H170K, Q90K+T105K+Q120K, Q90K+T105K+T133K,Q90K+T105K+S159K, Q90K+T105K+H170K, Q90K+Q120K+T133K, Q90K+Q120K+S159K,Q90K+Q120K+H170K, Q90K+T133K+S159K, Q90K+T133K+H170K, Q90+S159K+H170K,T105K+Q120K+T133K, T105K+Q120K+S159K, T105K+Q120K+H170K,T105K+T133K+S159K, T105K+T133K+H170K, T105K+S159K+H170K,Q120K+T133K+S159K, Q120K+T133K+H170K, Q120K+S159K+H170K,T133K+S159K+H170K, Q70K+Q90K+T105K+Q120K, Q70K+Q90K+T105K+T133K,Q70K+Q90K+T105K+S159K, Q70K+Q90K+T105K+H170K, Q70K+Q90K+Q120K+T133K,Q70K+Q90K+Q120K+S159K, Q70K+Q90K+Q120K+H170K, Q70K+Q90K+T133K+S159K,Q70K+Q90K+T133K+H170K, Q70K+Q90K+S159K+H170K, Q70K+T105K+Q120K+T133K,Q70K+T105K+Q120K+S159K, Q70K+T105K+Q120K+H170K, Q70K+T105K+T133K+S159K,Q70K+T105K+T133K+H170K, Q70K+T105K+S159K+H170K, Q70K+Q120K+T133K+S159K,Q70K+Q120K+T133K+H170K, Q70K+T133K+S159K+H170K, Q90K+T105K+Q120K+T133K,Q90K+T105K+Q120K+S159K, Q90K+T105K+Q120K+H170K, Q90K+T105+T133K+S159K,Q90K+T105+T133K+H170K, Q90K+T105+S159K+H170K, Q90K+Q120K+T133K+S159K,Q90K+Q120K+T133K+H170K, Q90K+Q120K+S159K+H170K, Q90K+T133K+S159K+H170K,T105K+Q120K+T133K+S159K, T105K+Q120K+T133K+H170K,T105K+Q120K+S159K+H170K, T105K+T133K+S159K+H170K orQ120K+T133K+S159K+H170K. In any of the variants listed above, thesubstitution H170K may instead be H170Q or H170R.

The polypeptide of the conjugate according to this aspect of theinvention, i.e. having at least one introduced and one removed lysine,preferably comprises at least one, such as one, two, three or four, ofthe substitutions selected from the group consisting of K16R, K16Q,K23R, K23Q, K34R, K34Q, K40R and K40Q, preferably at least thesubstitution K16R, whereby conjugation of this residue can be avoided.Preferably, the polypeptide comprises at least one substitution selectedfrom the group consisting of K16R+K23R, K16R+K34R, K16R+K40R, K23R+K34R,K23R+K40R, K34R+K40R, K16R+K23R+K34R, K16R+K23R+K40R, K23R+K34R+K40R,K16R+K34R+K40R and K16R+K23R+K34R+K40R. In one preferred embodiment, thepolypeptide includes the substitutions K16R+K34R+K40R, while the lysinein position 23 is left unaltered. As indicated above, it is contemplatedthat any of the individual substitutions or combinations listed in thisparagraph for removal of a lysine residue may suitably be used with anyof the other substitutions disclosed herein for introduction of lysineresidues, in particular the substitutions listed in the paragraph above.

In a particular embodiment the polypeptide includes the substitutionsK16R, K34R, K40R, T105K and S159K and is conjugated to 2-6, typically3-6 polyethylene glycol moieties with a molecular weight of about1000-10,000 Da.

In one embodiment the conjugate of the invention has a glycosylation inT133, i.e. this position is unaltered from the wild-type hG-CSF. This isthe natural glycosylation site. Alternatively, the conjugate may benon-glycosylated, although glycosylated conjugates are preferred.

In particular, the conjugate may have 2-6, typically 3-6 polyethyleneglycol moieties with a molecular weight of about 5000-6000 Da attached,e.g. mPEG with a molecular weight of about 5 kDa. Preferably, theconjugate has 4-5 polyethylene glycol moieties with a molecular weightof about 5000-6000 Da attached, e.g. 5 kDa mPEG.

In another embodiment, the conjugate may be produced so as to have onlya single number of PEG moieties attached, e.g. either 2, 3, 4, 5 or 6PEG moieties per polypeptide, or to have a desired mix of polypeptideconjugates with different numbers of PEG moieties attached, e.g. a mixhaving 2-5, 2-4, 3-5, 3-4, 4-6, 4-5 or 5-6 attached PEG moieties. Asindicated above, an example of a preferred conjugate mix is one having4-5 PEG moieties of about 5 kDa.

It will be understood that a conjugate having a specific number ofattached PEG moieties, or a mix of conjugates having a defined range ofnumbers of attached PEG moieties, may be obtained by choosing suitablePEGylation conditions and optionally by using subsequent purification toseparate conjugates having the desired number of PEG moieties. Examplesof methods for separation of G-CSF molecules with different numbers ofPEG moieties attached are provided below. Determination of the number ofattached PEG moieties may e.g. be performed using SDS-PAGE. For purposesof the present invention, a polypeptide conjugate may be considered tohave a given number of attached PEG moieties if separation on anSDS-PAGE gel shows no or only insignificant bands other than the band(s)corresponding to the given number(s) of PEG moieties. For example, asample of a polypeptide conjugate is considered to have 4-5 attached PEGgroups if an SDS-PAGE gel on which the sample has been run shows bandscorresponding to 4 and 5 PEG groups, respectively, and onlyinsignificant bands or, preferably, no bands corresponding to 3 or 6 PEGgroups.

While the non-polypeptide moiety of the conjugate according to thisaspect of the invention may be any molecule which, when using the givenconjugation method has lysine as an attachment group such as acarbohydrate moiety, it is preferred that the non-polypeptide moiety isa polymer molecule. The polymer molecule may be any of the moleculesmentioned in the section entitled “Conjugation to a polymer molecule”,but is preferably selected from the group consisting of linear orbranched polyethylene glycol or another polyalkylene oxide. Preferredpolymer molecules are e.g. mPEG-SPA (in particular SPA-mPEG 5000) fromShearwater Corp. or oxycarbonyl-oxy-N-dicarboxyimide PEG (U.S. Pat. No.5,122,614).

It will be understood that any of the amino acid changes, in particularsubstitutions, specified in this section can be combined with any of theamino acid changes, preferably substitutions, specified in the othersections herein disclosing specific amino acid modifications, includingintroduction and/or removal of glycosylation sites.

Conjugate of the Invention Wherein the Non-Polypeptide Moiety is aMolecule which has Cysteine as an Attachment Group

In another aspect the invention relates to a conjugate comprising

-   i) a polypeptide exhibiting G-CSF activity, which comprises an amino    acid sequence that differs from the amino acid sequence of hG-CSF    shown in SEQ ID NO:1 in at least one substitution selected from the    group consisting of T1C, P2C, L3C, G4C, P5C, A6C, S7C, S8C, L9C,    P10C, Q11C, S12C, F13C, L14C, L15C, E19C, Q20C, V21C, R22C, Q25C,    G26C, D27C, A29C, A30C, E33C, A37C, T38C, Y39C, L41C, H43C, P44C,    E45C, E46C, V48C, L49C, L50C, H52C, S53C, L54C, I56C, P57C, P60C,    L61C, S62C, S63C, P65C, S66C, Q67C, A68C, L69C, Q70C, L71C, A72C,    G73C, S76C, Q77C, L78C, S80C, F83C, Q86C, G87C, Q90C, E93C, G94C,    S96C, P97C, E98C, L99C, G100C, P101C, T102C, D104C, T105C, Q107C,    L108C, D109C, A111C, D112C, F113C, T115C, T116C, W118C, Q119C,    Q120C, M121C, E122C, E123C, L124C, M126C, A127C, P128C, A129C,    L130C, Q131C, P132C, T133C, Q134C, G135C, A136C, M137C, P138C,    A139C, A141C, S142C, A143C, F144C, Q145C, R146C, R147C, S155C,    H156C, Q158C, S159C, L161C, E162C, V163C, S164C, Y165C, R166C,    V167C, L168C, R169C, H170C, L171C, A172C, Q173C and P174C, and-   ii) at least one non-polypeptide moiety attached to a cysteine    residue of the polypeptide.

The receptor-binding domain of hG-CSF contains a cysteine residue inposition 17 which does not take part in a cystine and which mayadvantageously be removed in order to avoid conjugation of anon-polypeptide moiety to said cysteine. Accordingly, in another, morepreferred aspect the invention relates to a conjugate comprising

-   i) a polypeptide exhibiting G-CSF activity, which comprises an amino    acid sequence that differs from the amino acid sequence shown in SEQ    ID NO:1 in at least one substitution selected from the group    consisting of T1C, P2C, L3C, G4C, P5C, A6C, S7C, S8C, L9C, P10C,    Q11C, S12C, F13C, L14C, L15C, E19C, Q20C, V21C, R22C, Q25C, G26C,    D27C, A29C, A30C, E33C, A37C, T38C, Y39C, L41C, H43C, P44C, E45C,    E46C, V48C, L49C, L50C, H52C, S53C, L54C, I56C, P57C, P60C, L61C,    S62C, S63C, P65C, S66C, Q67C, A68C, L69C, Q70C, L71C, A72C, G73C,    S76C, Q77C, L78C, S80C, F83C, Q86C, G87C, Q90C, E93C, G94C, S96C,    P97C, E98C, L99C, G100C, P101C, T102C, D104C, T105C, Q107C, L108C,    D109C, A111C, D112C, F113C, T115C, T116C, W118C, Q119C, Q120C,    M121C, E122C, E123C, L124C, M126C, A127C, P128C, A129C, L130C,    Q131C, P132C, T133C, Q134C, G135C, A136C, M137C, P138C, A139C,    A141C, S142C, A143C, F144C, Q145C, R146C, R147C, S155C, H156C,    Q158C, S159C, L161C, E162C, V163C, S164C, Y165C, R166C, V167C,    L168C, R169C, H170C, L171C, A172C, Q173C and P174C, in combination    with removal of C17, preferably substitution of C17 with any other    amino acid residue, e.g. with a serine residue, and-   ii) a non-polypeptide moiety which has a cysteine residue as an    attachment group.

Preferred substitutions according to this aspect of the invention aresubstitutions of arginine with cysteine, for example one or more ofR146C, R147C, R166C and R169C.

It will be understood that any of the amino acid modifications, inparticular substitutions, specified in this section can be combined withany of the amino acid changes, in particular substitutions, specified inthe other sections herein disclosing specific amino acid modifications,including introduction and/or removal of glycosylation sites.

Conjugate of the Invention Wherein the Non-Polypeptide Moiety Binds toan Acid Group or the C-Terminal Amino Acid Residue

In a still further aspect the invention relates to a conjugatecomprising

-   i) a polypeptide exhibiting G-CSF activity, which comprises an amino    acid sequence that differs from the amino acid sequence shown in SEQ    ID NO:1 in at least one substitution selected from the group    consisting of T1D, P2D, L3D, G4D, P5D, A6D, S7D, S8D, L9D, P10D,    Q11D, S12D, F13D, L14D, L15D, K16D, Q20D, V21D, R22D, K23D, Q25D,    G26D, A29D, A30D, K34D, A37D, T38D, Y39D, K40D, L41D, H43D, P44D,    V48D, L49D, L50D, H52D, S53D, L54D, I56D, P57D, P60D, L61D, S62D,    S63D, P65D, S66D, Q67D, A68D, L69D, Q70D, L71D, A72D, G73D, S76D,    Q77D, L78D, S80D, F83D, Q86D, G87D, Q90D, G94D, S96D, P97D, L99D,    G100D, P101D, T102D, T105D, Q107D, L108D, A111D, F113D, T115D,    T116D, W118D, Q119D, Q120D, M121D, L124D, M126D, A127D, P128D,    A129D, L130D, Q131D, P132D, T133D, Q134D, G135D, A136D, M137D,    P138D, A139D, A141D, S142D, A143D, F144D, Q145D, R146D, R147D,    S155D, H156D, Q158D, S159D, L161D, V163D, S164D, Y165D, R166D,    V167D, L168D, R169D, H170D, L171D, A172D, Q173D and P174D; or at    least one substitution selected from the group consisting of T1E,    P2E, L3E, G4E, P5E, A6E, S7E, S8E, L9E, P10E, Q11E, S12E, F13E,    L14E, L15E, K16E, Q20E, V21E, R22E, K23E, Q25E, G26E, A29E, A30E,    K34E, A37E, T38E, Y39E, K40E, L41E, H43E, P44E, V48E, L49E, L50E,    H52E, S53E, L54E, I56E, P57E, P60E, L61E, S62E, S63E, P65E, S66E,    Q67E, A68E, L69E, Q70E, L71E, A72E, G73E, S76E, Q77E, L78E, S80E,    F83E, Q86E, G87E, Q90E, G94E, S96E, P97E, L99E, G100E, P101E, T102E,    T105E, Q107E, L108E, A111E, F113E, T115E, T116E, W118E, Q119E,    Q120E, M121E, L124E, M126E, A127E, P128E, A129E, L130E, Q131E,    P132E, T133E, Q134E, G135E, A136E, M137E, P138E, A139E, A141E,    S142E, A143E, F144E, Q145E, R146E, R147E, S155E, H156E, Q158E,    S159E, L161E, V163E, S164E, Y165E, R166E, V167E, L168E, R169E,    H170E, L171E, A172E, Q173E and P174E; and-   ii) a non-polypeptide moiety having an aspartic acid or a glutamic    acid residue as an attachment group.

Examples of preferred substitutions according to this aspect of theinvention include Q67D/E, Q70D/E, Q77D/E, Q86D/E, Q90D/E, Q120D/E,Q131D/E, Q134D/E, Q145D/E and Q173D/E.

In addition to the above listed substitutions, the polypeptide of theconjugate according to any of the above aspects may comprise removal,preferably by substitution, of at least one of the amino acid residuesselected from the group consisting of D27, D104, D109, D112, E19, E33,E45, E46, E93, E98, E122, E123, and E163. The substitution may be forany other amino acid residue, in particular for an asparagine or aglutamine residue, whereby conjugation of these residues can be avoided.In particular, the polypeptide may comprise at least one of thefollowing substitutions: D27N, D104N, D109N, D112N, E19Q, E33Q, E45Q,E46Q, E93Q, E98Q, E122Q, E123Q and E163Q. Preferably, the amino acidsubstitution in one or more of the above positions may in addition becombined with at least one of the following substitutions: D109N, D112N,E19Q, E122Q and E123Q.

While the non-polypeptide moiety of the conjugate according to thisaspect of the invention, which has an acid group as an attachment group,can be any non-polypeptide moiety with such property, it is presentlypreferred that the non-polypeptide moiety is a polymer molecule or anorganic derivatizing agent, in particular a polymer molecule, and theconjugate is prepared e.g. as described by Sakane and Pardridge,Pharmaceutical Research, Vol. 14, No. 8, 1997, pp 1085-1091.

It will be understood that any of the amino acid changes, in particularsubstitutions, specified in this section can be combined with any of theamino acid changes, in particular substitutions specified in the othersections herein disclosing specific amino acid changes, includingintroduction and/or removal of glycosylation sites.

Other Conjugates of the Invention

In addition to the non-polypeptide moieties specified above e.g. in thesections entitled “Conjugate of the invention . . . ” the conjugate ofthe invention may contain one or more carbohydrate moieties as aconsequence of the polypeptide being expressed in a glycosylating hostcell to result in glycosylation at the natural glycosylation site ofhG-CSF (T133) and/or at introduced glycosylation site(s).

Conjugate of the Invention Wherein the Non-Polypeptide Moiety is aCarbohydrate Moiety

In a further aspect the invention relates to a conjugate comprising aglycosylated polypeptide exhibiting G-CSF activity, which comprises anamino acid sequence that differs from that shown in SEQ ID NO:1 in thatat least one non-naturally occurring glycosylation site has beenintroduced into the amino acid sequence by way of at least onesubstitution selected from the group consisting of L3N+P5S/T, P5N, A6N,S8N+P10S/T, P10N, Q11N+F13S/T, S12N+L14S/T, F13N+L15S/T, L14N+K16S/T,K16N+L18S/T, E19N+V21S/T, Q20N+R22S/T, V21N+K23S/T, R22N+124S/T,K23N+Q25S/T, Q25N+D27S/T, G26N+G28S/T, D27N+A29S/T, A29N+L31S/T,A30N+Q32S/T, E33N+L35S/T, A37N+Y39S/T, T38N+K40S/T, Y39N+L41S/T,P44N+E46S/T, E45N+L47S/T, E46N+V48S/T, V48N+L50S/T, L49N+G51S/T,L50N+H52S/T, H52N+L54S/T, S53N+G55S/T, P60N, L61N, S63N+P65S/T,P65N+Q67S/T, S66N+A68S/T, Q67N+L69S/T, A68N+Q70S/T, L69N+L71S/T,Q70N+A72S/T, L71N+G73S/T, G73N+L75S/T, S76N+L78S/T, Q77N+H79S/T, L78N,S80N+L82S/T, F83N+Y85S/T, Q86N+L88S/T, G87N+L89S/T, Q90N+L92S/T,E93N+I95S/T, P97N+L99S/T, L99N+P101S/T, P101N+L103S/T, T102N+D104S/T,D104N+L106S/T, T105N+Q107S/T, Q107N+D109S/T, L108N+V110S/T,D109N+A111S/T, A111N+F113S/T, D112N+A114S/T, F113N, T115N+I117S/T,T116N+W118S/T, W118N+Q120S/T, Q119N+M121S/T, Q120N+E122S/T,M121N+E123S/T, E122N+L124S/T, E123N+G125S/T, L124N+M126S/T,M126N+P128S/T, P128N+L130S/T, L130N+P132S/T, P132N+Q134S/T,T133N+G135S/T, Q134N+A136S/T, A136N+P138S/T, P138N+F140S/T,A139N+A141S/T, A141N+A143S/T, S142N+F144S/T, A143N+Q145S/T,F144N+R146S/T, Q145N+R147S/T, R146N+A148S/T, R147N+G149S/T,S155N+L157S/T, H156N+Q158S/T, S159N+L161S/T, L161N+V163S/T, E162N,V163N+Y165S/T, S164N+R166S/T, Y165N+V167S/T, R166N+L168S/T,V167N+R169S/T, L168N+H170S/T, R169N+L171S/T and H170N+A172S/T, whereinS/T indicates an S or a T residue, preferably a T residue.

It will be understood that in order to prepare a conjugate according tothis aspect the polypeptide must be expressed in a glycosylating hostcell capable of attaching oligosaccharide moieties at the glycosylationsite(s) or alternatively subjected to in vitro glycosylation. Examplesof glycosylating host cells are given in the section further belowentitled “Coupling to an oligosaccharide moiety”.

Alternatively, the conjugate according to this aspect comprises apolypeptide exhibiting G-CSF activity, which comprises an amino acidsequence that differs from that shown in SEQ ID NO:1 in at least onesubstitution selected from the group consisting of P5N, A6N, P10N, P60N,L61N, L78N, F113N and E162N, in particular from the group consisting ofP5N, A6N, P10N, P60N, L61N, F113N and E162N, such as from the groupconsisting of P60N, L61N, F113N and E162N.

Alternatively, the conjugate according to this aspect comprises apolypeptide exhibiting G-CSF activity, which comprises an amino acidsequence that differs from that shown in SEQ ID NO:1 in at least onesubstitution selected from the group consisting of D27N+A29S, D27N+A29T,D104N+L106S, D104N+L106T, D109N+A111S, D109N+A111T, D112N+A114S andD112N+A114T, more preferably from the group consisting of D27N+A29S,D27N+A29T, D104N+L106S, D104N+L106T, D112N+A114S and D112N+A114T, suchas from the group consisting of D27N+A29S, D27N+A29T, D104N+L106S andD104N+L106T.

In addition to a carbohydrate molecule, the conjugate according to theaspect of the invention described in the present section may containadditional non-polypeptide moieties, in particular a polymer molecule,as described in the present application, conjugated to one or moreattachment groups present in the polypeptide part of the conjugate.

It will be understood that any of the amino acid changes, in particularsubstitutions, specified in this section can be combined with any of theamino acid changes, in particular substitutions, specified in the othersections herein disclosing specific amino acid changes.

Circularly Permuted Variants

In a further embodiment, the polypeptide part of the polypeptideconjugate of the invention may be in the form of a circularly permutedvariant of a polypeptide sequence otherwise disclosed herein. In such acircularly permuted polypeptide, the original N-terminus and C-terminusare joined together either directly by a peptide bond or indirectly viaa peptide linker, while new N- and C-termini are formed between twoadjacent amino acid residues that originally were joined by a peptidebond. Since the original N- and C-termini will normally be located atsome distance from each other, they will typically be linked by means ofa peptide linker having a suitable length and composition so that thestructure and activity of the conjugate is not adversely affected. Itwill be clear that the new N-terminus and C-terminus should not beformed between an amino acid residue pair where this would interferewith the activity of the polypeptide. Circularly permuted G-CSF receptoragonists are disclosed in U.S. Pat. No. 6,100,070, to which reference ismade for further information on selecting peptide linkers and thelocation of the new N-terminus and C-terminus as well as methods forproducing them such variants.

White Blood Cell and Neutrophil Formation of Conjugates of the Invention

In a further embodiment, the polypeptide conjugate of the invention maybe characterized as being a conjugate exhibiting G-CSF activity andcomprising a polypeptide with an amino acid sequence that differs in atleast one amino acid residue from the amino acid sequence shown in SEQID NO:1 and having at least one non-polypeptide moiety attached to anattachment group of the polypeptide, the polypeptide conjugate furtherfulfilling at least one of the following criteria (A)-(D):

(A) after one subcutaneous administration of 100 microgram per kg bodyweight to rats (based on the weight of the polypeptide part of theconjugate) it:

-   -   i) increases formation of white blood cells with at least about        the same rate and to at least about the same level (measured as        number of cells per liter of blood) as administration of 100        microgram of non-conjugated hG-CSF per kg body weight for a        period of 6 hours, preferably 12 hours after administration, and    -   ii) increases the level of white blood cells (measured as number        of cells per liter blood) above the level of white blood cells        prior to administration for a period of at least about 96 hours,        preferably for at least about 120 hours;

(B) after one subcutaneous administration of 25 microgram per kg bodyweight to rats (based on the weight of the polypeptide part of theconjugate) it:

-   -   i) increases formation of white blood cells with at least about        the same rate and to at least about the same level (measured as        number of cells per liter of blood) as administration of 100        microgram of non-conjugated hG-CSF per kg body weight for a        period of 6 hours, preferably 12 hours after administration, and    -   ii) increases the level of white blood cells (measured as number        of cells per liter blood) above the level of white blood cells        prior to administration for a period of at least about 72 hours,        preferably at least about 96 hours, more preferably at least        about 120 hours;

(C) after one subcutaneous administration of 100 microgram per kg bodyweight to rats (based on the weight of the polypeptide part of theconjugate) it:

-   -   i) increases formation of neutrophils with at least about the        same rate and to at least about the same level (measured as        number of cells per liter of blood) as administration of 100        microgram of non-conjugated hG-CSF per kg body weight for a        period of 6 hours, preferably 12 hours after administration, and    -   ii) increases the level of neutrophils (measured as number of        cells per liter blood) above the level of neutrophils prior to        administration for a period of at least about 96 hours,        preferably at least about 120 hours;

(D) after one subcutaneous administration of 25 microgram per kg bodyweight to rats (based on the weight of the polypeptide part of theconjugate) it:

-   -   i) increases formation of neutrophils with at least about the        same rate and to at least about the same level (measured as        number of cells per liter of blood) as administration of 100        microgram of non-conjugated hG-CSF per kg body weight for a        period of 6 hours, preferably 12 hours after administration, and    -   ii) increases the level of neutrophils (measured as number of        cells per liter blood) above the level of neutrophils prior to        administration for a period of at least about 72 hours,        preferably at least about 96 hours, more preferably at least        about 120 hours.        Non-Polypeptide Moiety of the Conjugate of the Invention

As indicated further above the non-polypeptide moiety of the conjugateof the invention is preferably selected from the group consisting of apolymer molecule, a lipophilic compound, a carbohydrate moiety (e.g. byway of in vivo glycosylation) and an organic derivatizing agent. All ofthese agents may confer desirable properties to the polypeptide part ofthe conjugate, in particular increased functional in vivo half-lifeand/or increased serum half-life. The polypeptide part of the conjugateis normally conjugated to only one type of non-polypeptide moiety, butmay also be conjugated to two or more different types of non-polypeptidemoieties, e.g. to a polymer molecule and an oligosaccharide moiety, to alipophilic group and an oligosaccharide moiety, to an organicderivatizing agent and an oligosaccharide moiety, to a lipophilic groupand a polymer molecule, etc. The conjugation to two or more differentnon-polypeptide moieties may be done simultaneously or sequentially.

Methods for Preparing a Conjugate of the Invention

In the following sections “Conjugation to a lipophilic compound”,“Conjugation to a polymer molecule”, “Conjugation to an oligosaccharidemoiety” and “Conjugation to an organic derivatizing agent” conjugationto specific types of non-polypeptide moieties is described. In general,a polypeptide conjugate according to the invention may be produced byculturing an appropriate host cell under conditions conducive forexpression of the polypeptide, and recovering the polypeptide, whereina) the polypeptide comprises at least one N- or O-glycosylation site andthe host cell is a eukaryotic host cell capable of in vivoglycosylation, and/or b) the polypeptide is subjected to conjugation toa non-polypeptide moiety in vitro.

Conjugation to a Lipophilic Compound

The polypeptide and the lipophilic compound may be conjugated to eachother, either directly or by use of a linker. The lipophilic compoundmay be a natural compound such as a saturated or unsaturated fatty acid,a fatty acid diketone, a terpene, a prostaglandin, a vitamin, acarotenoid or steroid, or a synthetic compound such as a carbon acid, analcohol, an amine and sulphonic acid with one or more alkyl, aryl,alkenyl or other multiple unsaturated compounds. The conjugation betweenthe polypeptide and the lipophilic compound, optionally through alinker, may be done according to methods known in the art, e.g. asdescribed by Bodanszky in Peptide Synthesis, John Wiley, New York, 1976and in WO 96/12505.

Conjugation to a Polymer Molecule

The polymer molecule to be coupled to the polypeptide may be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror heteropolymer, typically with a molecular weight in the range ofabout 300-100,000 Da, such as about 500-20,000 Da, more preferably inthe range of about 1000-15,000 Da, even more preferably in the range ofabout 2000-12,000 Da, such as about 3000-10,000. When used about polymermolecules herein, the word “about” indicates an approximate averagemolecular weight and reflects the fact that there will normally be acertain molecular weight distribution in a given polymer preparation.

Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine(i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Ahetero-polymer is a polymer which comprises different coupling groups,such as a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as linear or branchedpolyethylene glycol (PEG) and polypropylene glycol (PPG), poly-vinylalcohol (PVA), poly-carboxylate, poly-(vinylpyrolidone),polyethylene-co-maleic acid anhydride, polystyrene-co-maleic acidanhydride, dextran, including carboxymethyl-dextran, or any otherbiopolymer suitable for reducing immunogenicity and/or increasingfunctional in vivo half-life and/or serum half-life. Another example ofa polymer molecule is human albumin or another abundant plasma protein.Generally, polyalkylene glycol-derived polymers are biocompatible,non-toxic, non-antigenic, non-immunogenic, have various water solubilityproperties, and are easily excreted from living organisms.

PEG is the preferred polymer molecule, since it has only few reactivegroups capable of cross-linking compared to polysaccharides such asdextran. In particular, monofunctional PEG, e.g. methoxypolyethyleneglycol (mPEG), is of interest since its coupling chemistry is relativelysimple (only one reactive group is available for conjugating withattachment groups on the polypeptide). Consequently, the risk ofcross-linking is eliminated, the resulting polypeptide conjugates aremore homogeneous and the reaction of the polymer molecules with thepolypeptide is easier to control.

To effect covalent attachment of the polymer molecule(s) to thepolypeptide, the hydroxyl end groups of the polymer molecule areprovided in activated form, i.e. with reactive functional groups.Suitable activated polymer molecules are commercially available, e.g.from Shearwater Corp., Huntsville, Ala., USA, or from PolyMASCPharmaceuticals plc, UK. Alternatively, the polymer molecules can beactivated by conventional methods known in the art, e.g. as disclosed inWO 90/13540. Specific examples of activated linear or branched polymermolecules for use in the present invention are described in theShearwater Corp. 1997 and 2000 Catalogs (Functionalized BiocompatiblePolymers for Research and pharmaceuticals, Polyethylene Glycol andDerivatives, incorporated herein by reference). Specific examples ofactivated PEG polymers include the following linear PEGs: NHS-PEG (e.g.SPA-PEG, SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, andSCM-PEG), and NOR-PEG), BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG,ALD-PEG, TRES-PEG, VS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs suchas PEG2-NHS and those disclosed in U.S. Pat. No. 5,932,462 and U.S. Pat.No. 5,643,575, both of which are incorporated herein by reference.Furthermore, the following publications, incorporated herein byreference, disclose useful polymer molecules and/or PEGylationchemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,476,653, WO97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No.5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No.5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No. 5,516,673,EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400 472, EP 183 503and EP 154 316.

The conjugation of the polypeptide and the activated polymer moleculesis conducted by use of any conventional method, e.g. as described in thefollowing references (which also describe suitable methods foractivation of polymer molecules): R. F. Taylor, (1991), “Proteinimmobilisation. Fundamental and applications”, Marcel Dekker, N.Y.; S.S. Wong, (1992), “Chemistry of Protein Conjugation and Crosslinking”,CRC Press, Boca Raton; G. T. Hermanson et al., (1993), “ImmobilizedAffinity Ligand Techniques”, Academic Press, N.Y.). The skilled personwill be aware that the activation method and/or conjugation chemistry tobe used depends on the attachment group(s) of the polypeptide (examplesof which are given further above), as well as the functional groups ofthe polymer (e.g. being amine, hydroxyl, carboxyl, aldehyde, sulfydryl,succinimidyl, maleimide, vinysulfone or haloacetate). The PEGylation maybe directed towards conjugation to all available attachment groups onthe polypeptide (i.e. such attachment groups that are exposed at thesurface of the polypeptide) or may be directed towards one or morespecific attachment groups, e.g. the N-terminal amino group (U.S. Pat.No. 5,985,265). Furthermore, the conjugation may be achieved in one stepor in a stepwise manner (e.g. as described in WO 99/55377).

It will be understood that the PEGylation is designed so as to producethe optimal molecule with respect to the number of PEG moleculesattached, the size and form of such molecules (e.g. whether they arelinear or branched), and where in the polypeptide such molecules areattached. The molecular weight of the polymer to be used will be chosentaking into consideration the desired effect to be achieved. Forinstance, if the primary purpose of the conjugation is to achieve aconjugate having a high molecular weight and larger size (e.g. to reducerenal clearance), one may choose to conjugate either one or a few highmolecular weight polymer molecules or a number of polymer molecules witha smaller molecular weight to obtain the desired effect. Preferably,however, several polymer molecules with a lower molecular weight will beused. This is also the case if a high degree of epitope shielding isdesired. In such cases, 2-8 polymers with a molecular weight of e.g.about 5,000 Da, such as 3-6 such polymers, may for example be used. Asthe examples below illustrate, it may be advantageous to have a largernumber of polymer molecules with a lower molecular weight (e.g. 4-6 witha MW of 5000) compared to a smaller number of polymer molecules with ahigher molecular weight (e.g. 1-3 with a MW of 12,000-20,000) in termsof improving the functional in vivo half-life of the polypeptideconjugate, even where the total molecular weight of the attached polymermolecules in the two cases is the same or similar. It is believed thatthe presence of a larger number of smaller polymer molecules providesthe polypeptide with a larger diameter or apparent size than e.g. asingle yet larger polymer molecule, at least when the polymer moleculesare relatively uniformly distributed on the polypeptide surface.

It has further been found that advantageous results are obtained whenthe apparent size (also referred to as the “apparent molecular weight”or “apparent mass”) of at least a major portion of the conjugate of theinvention is at least about 50 kDa, preferably at least about 55 kDa,more preferably at least about 60 kDa, e.g. at least about 66 kDa. Thisis believed to be due to the fact that renal clearance is substantiallyeliminated for conjugates having a sufficiently large apparent size. Inthe present context, the “apparent size” of a G-CSF conjugate orpolypeptide is determined by the SDS-PAGE method described in theexamples section below.

The use of the term “major portion” is related to the fact that thepolypeptide conjugates of the invention will typically compriseindividual conjugates having varying numbers of non-polypeptide moietiesattached. For example, a given polypeptide subjected to PEGylation undera given set of PEGylation conditions may result in a composition inwhich most of the individual polypeptide conjugates have e.g. between 3and 5 PEG groups attached, with a majority of the conjugates having 4PEG groups attached. It will be clear that the apparent molecular weightof these individual conjugate molecules will vary. In this example, ifwe assume that a G-CSF polypeptide is conjugated to PEG groups with a MWof 5 kDa, conjugates having only 3 PEG groups attached will be seen onan SDS-PAGE gel as a band that is likely to have an apparent molecularweight of less than about 50 kDa, while conjugates having 4 or 5 PEGgroups attached will result in bands with progressively higher apparentmolecular weights that most likely all are greater than about 50 kDa.Thus, in this example there would be 3 major bands on an SDS-PAGE gel,corresponding to conjugates with 3, 4 or 5 attached PEG groups,respectively. The term “major portion” in the context of the presentspecification and claims is therefore intended to refer to the fact thatat least one of these major bands on an SDS-PAGE gel will correspond tothe indicated minimum apparent molecular weight.

Preferably, at least 50% of the individual conjugate molecules will havea minimum apparent size as described above. More preferably, at least60% of the individual conjugate molecules with have such a minimumapparent size, still more preferably at least 70%, 75%, 80% or 85%. Mostpreferably, at least 90% of the individual conjugate molecules will havea minimum apparent size as described above, i.e. at least 50 kDa andpreferably higher, such as at least 55 kDa or 60 kDa.

It will be understood that the apparent size in kDa of a conjugate orpolypeptide is not necessarily the same as the actual molecular weightof the conjugate or polypeptide. Rather, the apparent size is areflection of both the actual molecular weight and the overall bulk.Since, in most cases, attachment of one or more PEG groups or othernon-polypeptide moieties will result in a relatively large increase ofthe bulk of the polypeptide to which such moieties are attached, thepolypeptide conjugates of the invention will normally have an apparentsize that exceeds the actual molecular weight of the conjugate.Therefore, in connection with renal clearance, a conjugate of theinvention can easily exhibit properties characteristic of a polypeptidewith a molecular weight above e.g. 66 kDa (corresponding to the apparentsize) but have an actual molecular weight well below 66 kDa. This effecton apparent size is believed to be responsible for the observation thatattachment of, for example, four PEG groups each having a molecularweight of 5 kDa provides results that are superior to a correspondingpolypeptide with a single 20 kDa PEG group attached.

While conjugation of only a single polymer molecule to a singleattachment group on the protein is not preferred, in the event that onlyone polymer molecule is attached, it will generally be advantageous thatthe polymer molecule, which may be linear or branched, has a relativelyhigh molecular weight, e.g. about 20 kDa.

In a further preferred embodiment, the conjugates of the inventionhave 1) at least a major portion with an apparent molecular weight of atleast about 50 kDa and 2) a reduced in vitjo bioactivity (reducedreceptor binding affinity) compared to hG-CSF as described above. It hasbeen found that such conjugates have both a low renal clearance as aresult of the large apparent size and a low receptor-mediated clearanceas a result of the low in vitro bioactivity (low receptor bindingaffinity). The overall result is excellent performance in terms ofeffective stimulation of neutrophils together with a significantlyincreased in vivo half-life and thus a long duration of action thatprovides important clinical advantages.

Normally, the polymer conjugation is performed under conditions aimingat reacting as many of the available polymer attachment groups aspossible with polymer molecules. This is achieved by means of a suitablemolar excess of the polymer in relation to the polypeptide (number ofattachment sites). Typical molar ratios of activated polymer moleculesto polypeptide attachment sites are up to about 1000-1, such as up toabout 200-1 or up to about 100-1. In some cases, the ratio may besomewhat lower, however, such as up to about 50-1, 10-1 or 5-1, e.g. ifa lower degree of polymer attachment is desired.

It is also contemplated according to the invention to couple the polymermolecules to the polypeptide through a linker. Suitable linkers are wellknown to the skilled person. A preferred example is cyanuric chloride(Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat.No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed.,24, 375-378.

Subsequent to the conjugation residual activated polymer molecules areblocked according to methods known in the art, e.g. by addition ofprimary amine to the reaction mixture, and the resulting inactivatedpolymer molecules are removed by a suitable method (see Materials andMethods).

In a preferred embodiment, the polypeptide conjugate of the inventioncomprises a PEG molecule attached to some, most or preferablysubstantially all of the lysine residues in the polypeptide availablefor PEGylation, in particular a linear or branched PEG molecule, e.g.with a molecular weight of about 1-15 kDa, typically about 2-12 kDa,such as about 3-10 kDa, e.g. about 5 or 6 kDa.

It will be understood that depending on the circumstances, e.g. theamino acid sequence of the polypeptide, the nature of the activated PEGcompound being used and the specific PEGylation conditions, includingthe molar ratio of PEG to polypeptide, varying degrees of PEGylation maybe obtained, with a higher degree of PEGylation generally being obtainedwith a higher ratio of PEG to polypeptide. The PEGylated polypeptidesresulting from any given PEGylation process will, however, normallycomprise a stochastic distribution of polypeptide conjugates havingslightly different degrees of PEGylation. If desired, such a mixture ofpolypeptide species having different numbers of PEG moieties attachedmay be subjected to purification, e.g. using the methods described inthe examples below, to obtain a product having a more uniform degree ofPEGylation.

In yet another embodiment, the polypeptide conjugate of the inventionmay comprise a PEG molecule attached to the lysine residues in thepolypeptide available for PEGylation, and in addition to the N-terminalamino acid residue of the polypeptide.

Coupling to an Oligosaccharide Moiety

The conjugation to an oligosaccharide moiety may take place in vivo orin vitro. In order to achieve in vivo glycosylation of a G-CSF moleculecomprising one or more glycosylation sites the nucleotide sequenceencoding the polypeptide must be inserted in a glycosylating, eukaryoticexpression host. The expression host cell may be selected from fungal(filamentous fungal or yeast), insect or animal cells or from transgenicplant cells. In one embodiment the host cell is a mammalian cell, suchas a CHO cell, BHK or HEK, e.g. HEK 293, cell, or an insect cell, suchas an SF9 cell, or a yeast cell, e.g. S. cerevisiae or Pichia pastoris,or any of the host cells mentioned hereinafter. Covalent in vitrocoupling of glycosides (such as dextran) to amino acid residues of thepolypeptide may also be used, e.g. as described in WO 87/05330 and inAplin et al., CRC Crit Rev. Biochem., pp. 259-306, 1981.

The in vitro coupling of oligosaccharide moieties or PEG to protein- andpeptide-bound Gln-residues can be carried out by transglutaminases(TG'ases). Transglutaminases catalyze the transfer of donor amine-groupsto protein- and peptide-bound Gln-residues in a so-called cross-linkingreaction. The donor-amine groups can be protein- or peptide-bound e.g.as the ε-amino-group in Lys-residues or can be part of a small or largeorganic molecule. An example of a small organic molecule functioning asan amino-donor in TG'ase-catalyzed cross-linking is putrescine(1,4-diaminobutane). An example of a larger organic molecule functioningas an amino-donor in TG'ase-catalyzed cross-linking is anamine-containing PEG (Sato et al., Biochemistry 35, 13072-13080).

TG'ases are in general highly specific enzymes, and not everyGln-residue exposed on the surface of a protein is accessible toTG'ase-catalyzed cross-linking to amino-containing substances. On thecontrary, only a few Gln-residues function naturally as TG'asesubstrates, but the exact parameters governing which Gln-residues aregood TG'ase substrates remain unknown. Thus, in order to render aprotein susceptible to TG'ase-catalyzed cross-linking reactions it isoften a prerequisite to add at convenient positions stretches of aminoacid sequence known to function very well as TG'ase substrates. Severalamino acid sequences are known to be or to contain excellent naturalTG'ase substrates e.g. substance P, elafin, fibrinogen, fibronectin,α₂-plasmin inhibitor, α-caseins, and β-caseins.

Coupling to an Organic Derivatizing Agent

Covalent modification of the polypeptide exhibiting G-CSF activity maybe performed by reacting one or more attachment groups of thepolypeptide with an organic derivatizing agent. Suitable derivatizingagents and methods are well known in the art. For example, cysteinylresidues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(4-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole. Histidyl residues are derivatizedby reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent isrelatively specific for the histidyl side chain. Para-bromophenacylbromide is also useful. The reaction is preferably performed in 0.1 Msodium cacodylate at pH 6.0. Lysinyl and amino terminal residues arereacted with succinic or other carboxylic acid anhydrides.Derivatization with these agents has the effect of reversing the chargeof the lysinyl residues. Other suitable reagents for derivatizingα-amino-containing residues include imidoesters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione andtransaminase-catalyzed reaction with glyoxylate. Arginyl residues aremodified by reaction with one or several conventional reagents, amongthem phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, andninhydrin. Derivatization of arginine residues requires that thereaction be performed in alkaline conditions because of the high pKa ofthe guanidine functional group.

Furthermore, these reagents may react with the groups of lysine as wellas the arginine guanidino group. Carboxyl side groups (aspartyl orglutamyl) are selectively modified by reaction with carbodiimides(R—N═C═N—R′), where R and R′ are different alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Blocking of the Functional Site

It has been reported that excessive polymer conjugation can lead to aloss of activity of the polypeptide to which the polymer is conjugated.This problem can be eliminated by e.g. removal of attachment groupslocated at the functional site or by blocking the functional site priorto conjugation so that the functional site is blocked duringconjugation. The latter strategy constitutes a further embodiment of theinvention (the first strategy being exemplified further above, e.g. byremoval of lysine residues which may be located close to the functionalsite). More specifically, according to the second strategy theconjugation between the polypeptide and the non-polypeptide moiety isconducted under conditions where the functional site of the polypeptideis blocked by a helper molecule capable of binding to the functionalsite of the polypeptide.

Preferably, the helper molecule is one which specifically recognizes afunctional site of the polypeptide, such as a receptor, in particularthe G-CSF receptor or a part of the G-CSF receptor.

Alternatively, the helper molecule may be an antibody, in particular amonoclonal antibody recognizing the polypeptide exhibiting G-CSFactivity. In particular, the helper molecule may be a neutralizingmonoclonal antibody.

The polypeptide is allowed to interact with the helper molecule beforeeffecting conjugation. This ensures that the functional site of thepolypeptide is shielded or protected and consequently unavailable forderivatization by the non-polypeptide moiety such as a polymer.Following its elution from the helper molecule, the conjugate betweenthe non-polypeptide moiety and the polypeptide can be recovered with atleast a partially preserved functional site.

The subsequent conjugation of the polypeptide having a blockedfunctional site to a polymer, a lipophilic compound, an oligosaccharidemoiety, an organic derivatizing agent or any other compound is conductedin the normal way, e.g. as described in the sections above entitled“Conjugation to . . . ”.

Irrespective of the nature of the helper molecule to be used to shieldthe functional site of the polypeptide from conjugation, it is desirablethat the helper molecule is free of or comprises only a few attachmentgroups for the non-polypeptide moiety of choice in part(s) of themolecule where the conjugation to such groups would hamper desorption ofthe conjugated polypeptide from the helper molecule. Hereby, selectiveconjugation to attachment groups present in non-shielded parts of thepolypeptide can be obtained and it is possible to reuse the helpermolecule for repeated cycles of conjugation. For instance, if thenon-polypeptide moiety is a polymer molecule such as PEG, which has theepsilon amino group of a lysine or N-terminal amino acid residue as anattachment group, it is desirable that the helper molecule issubstantially free of conjugatable epsilon amino groups, preferably freeof any epsilon amino groups. Accordingly, in a preferred embodiment thehelper molecule is a protein or peptide capable of binding to thefunctional site of the polypeptide, which protein or peptide is free ofany conjugatable attachment groups for the non-polypeptide moiety ofchoice.

Of particular interest in connection with the embodiment of the presentinvention wherein the polypeptide conjugates are prepared from adiversified population of nucleotide sequences encoding a polypeptide ofinterest, the blocking of the functional group is effected in microtiterplates prior to conjugation, for instance by plating the expressedpolypeptide variant in a microtiter plate containing an immobilizedblocking group such as a receptor, an antibody or the like.

In a further embodiment the helper molecule is first covalently linkedto a solid phase such as column packing materials, for instance Sephadexor agarose beads, or a surface, e.g. a reaction vessel. Subsequently,the polypeptide is loaded onto the column material carrying the helpermolecule and conjugation carried out according to methods known in theart, e.g. as described in the sections above entitled “Conjugation to .. . ”. This procedure allows the polypeptide conjugate to be separatedfrom the helper molecule by elution. The polypeptide conjugate is elutedby conventional techniques under physico-chemical conditions that do notlead to a substantive degradation of the polypeptide conjugate. Thefluid phase containing the polypeptide conjugate is separated from thesolid phase to which the helper molecule remains covalently linked. Theseparation can be achieved in other ways: For instance, the helpermolecule may be derivatized with a second molecule (e.g. biotin) thatcan be recognized by a specific binder (e.g. streptavidin). The specificbinder may be linked to a solid phase, thereby allowing the separationof the polypeptide conjugate from the helper molecule-second moleculecomplex through passage over a second helper-solid phase column whichwill retain, upon subsequent elution, the helper molecule-secondmolecule complex, but not the polypeptide conjugate. The polypeptideconjugate may be released from the helper molecule in any appropriatefashion. Deprotection may be achieved by providing conditions in whichthe helper molecule dissociates from the functional site of the G-CSF towhich it is bound. For instance, a complex between an antibody to whicha polymer is conjugated and an anti-idiotypic antibody can bedissociated by adjusting the pH to an acid or alkaline pH.

Conjugation of a Tagged Polypeptide

In an alternative embodiment the polypeptide is expressed as a fusionprotein with a tag, i.e. an amino acid sequence or peptide stretch madeup of typically 1-30, such as 1-20 amino acid residues. Besides allowingfor fast and easy purification, the tag is a convenient tool forachieving conjugation between the tagged polypeptide and thenon-polypeptide moiety. In particular, the tag may be used for achievingconjugation in microtiter plates or other carriers, such as paramagneticbeads, to which the tagged polypeptide can be immobilized via the tag.The conjugation to the tagged polypeptide in e.g. microtiter plates hasthe advantage that the tagged polypeptide can be immobilized in themicrotiter plates directly from the culture broth (in principle withoutany purification) and subjected to conjugation. Thereby, the totalnumber of process steps (from expression to conjugation) can be reduced.Furthermore, the tag may function as a spacer molecule, ensuring animproved accessibility to the immobilized polypeptide to be conjugated.The conjugation using a tagged polypeptide may be to any of thenon-polypeptide moieties disclosed herein, e.g. to a polymer moleculesuch as PEG.

The identity of the specific tag to be used is not critical as long asthe tag is capable of being expressed with the polypeptide and iscapable of being immobilized on a suitable surface or carrier material.A number of suitable tags are commercially available, e.g. from UnizymeLaboratories, Denmark. For instance, the tag may consist of any of thefollowing sequences:

(SEQ ID NO:5) Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-Gln (SEQ ID NO:9) His-His-His-His-His-His (SEQ ID NO:10)Met-Lys-His-His-His-His-His-His (SEQ ID NO:11)Met-Lys-His-His-Ala-His-His-Gln-His-His (SEQ ID NO:12)Met-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln- His-Glnor any of the following:

-   EQKLI SEEDL (SEQ ID NO:13; a C-terminal tag described in Mol. Cell.    Biol. 5:3610-16, 1985) DYKDDDDK (SEQ ID NO:14; a C- or N-terminal    tag) YPYDVPDYA (SEQ ID NO:15)

Antibodies against the above tags are commercially available, e.g. fromADI, Aves Lab and Research Diagnostics.

A convenient method for using a tagged polypeptide for PEGylation isgiven in the Materials and Methods section below. The subsequentcleavage of the tag from the polypeptide may be achieved by use ofcommercially available enzymes.

Methods for Preparing a Polypeptide of the Invention or the PolypeptidePart of the Conjugate of the Invention

The polypeptide of the present invention or the polypeptide part of aconjugate of the invention, optionally in glycosylated form, may beproduced by any suitable method known in the art. Such methods includeconstructing a nucleotide sequence encoding the polypeptide andexpressing the sequence in a suitable transformed or transfected host.However, polypeptides of the invention may be produced, albeit lessefficiently, by chemical synthesis or a combination of chemicalsynthesis or a combination of chemical synthesis and recombinant DNAtechnology.

A nucleotide sequence encoding a polypeptide or the polypeptide part ofa conjugate of the invention may be constructed by isolating orsynthesizing a nucleotide sequence encoding the parent G-CSF, such ashG-CSF with the amino acid sequence shown in SEQ ID NO:1, and thenchanging the nucleotide sequence so as to effect introduction (i.e.insertion or substitution) or deletion (i.e. removal or substitution) ofthe relevant amino acid residue(s).

The nucleotide sequence is conveniently modified by site-directedmutagenesis in accordance with conventional methods. Alternatively, thenucleotide sequence is prepared by chemical synthesis, e.g. by using anoligonucleotide synthesizer, wherein oligonucleotides are designed basedon the amino acid sequence of the desired polypeptide, and preferablyselecting those codons that are favored in the host cell in which therecombinant polypeptide will be produced. For example, several smalloligonucleotides coding for portions of the desired polypeptide may besynthesized and assembled by PCR, ligation or ligation chain reaction(LCR) (Barany, PNAS 88:189-193, 1991). The individual oligonucleotidestypically contain 5′ or 3′ overhangs for complementary assembly.

Alternative nucleotide sequence modification methods are available forproducing polypeptide variants for high throughput screening, forinstance methods which involve homologous cross-over such as disclosedin U.S. Pat. No. 5,093,257, and methods which involve gene shuffling,i.e. recombination between two or more homologous nucleotide sequencesresulting in new nucleotide sequences having a number of nucleotidealterations when compared to the starting nucleotide sequences. Geneshuffling (also known as DNA shuffling) involves one or more cycles ofrandom fragmentation and reassembly of the nucleotide sequences,followed by screening to select nucleotide sequences encodingpolypeptides with desired properties. In order for homology-basednucleic acid shuffling to take place, the relevant parts of thenucleotide sequences are preferably at least 50% identical, such as atleast 60% identical, more preferably at least 70% identical, such as atleast 80% identical. The recombination can be performed in vitro or invivo.

Examples of suitable in vitro gene shuffling methods are disclosed byStemmer et al. (1994), Proc. Natl. Acad. Sci. USA; vol. 91, pp.10747-10751; Stemmer (1994), Nature, vol. 370, pp. 389-391; Smith(1994), Nature vol. 370, pp. 324-325; Zhao et al., Nat. Biotechnol.1998, March; 16(3): 258-61; Zhao H. and Arnold, FB, Nucleic AcidsResearch, 1997, Vol. 25. No. 6 pp. 1307-1308; Shao et al., Nucleic AcidsResearch 1998, Jan. 15; 26(2): pp. 681-83; and WO 95/17413. An exampleof a suitable in vivo shuffling method is disclosed in WO 97/07205.Other techniques for mutagenesis of nucleic acid sequences by in vitroor in vivo recombination are disclosed e.g. in WO 97/20078 and U.S. Pat.No. 5,837,458. Examples of specific shuffling techniques include “familyshuffling”, “synthetic shuffling” and “in silico shuffling”. Familyshuffling involves subjecting a family of homologous genes fromdifferent species to one or more cycles of shuffling and subsequentscreening or selection. Family shuffling techniques are disclosed e.g.by Crameri et al. (1998), Nature, vol. 391, pp. 288-291; Christians etal. (1999), Nature Biotechnology, vol. 17, pp. 259-264; Chang et al.(1999), Nature Biotechnology, vol. 17, pp. 793-797; and Ness et al.(1999), Nature Biotechnology, vol. 17, 893-896. Synthetic shufflinginvolves providing libraries of overlapping synthetic oligonucleotidesbased e.g. on a sequence alignment of homologous genes of interest. Thesynthetically generated oligonucleotides are recombined, and theresulting recombinant nucleic acid sequences are screened and if desiredused for further shuffling cycles. Synthetic shuffling techniques aredisclosed in WO 00/42561. In silico shuffling refers to a DNA shufflingprocedure which is performed or modelled using a computer system,thereby partly or entirely avoiding the need for physically manipulatingnucleic acids. Techniques for in silico shuffling are disclosed in WO00/42560.

Once assembled (by synthesis, site-directed mutagenesis or anothermethod), the nucleotide sequence encoding the polypeptide is insertedinto a recombinant vector and operably linked to control sequencesnecessary for expression of the G-CSF in the desired transformed hostcell.

It should of course be understood that not all vectors and expressioncontrol sequences function equally well to express the nucleotidesequence encoding a polypeptide described herein. Neither will all hostsfunction equally well with the same expression system. However, one ofskill in the art may make a selection among these vectors, expressioncontrol sequences and hosts without undue experimentation. For example,in selecting a vector, the host must be considered because the vectormust replicate in it or be able to integrate into the chromosome. Thevector's copy number, the ability to control that copy number, and theexpression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered. In selecting anexpression control sequence, a variety of factors should also beconsidered. These include, for example, the relative strength of thesequence, its controllability, and its compatibility with the nucleotidesequence encoding the polypeptide, particularly as regards potentialsecondary structures. Hosts should be selected by consideration of theircompatibility with the chosen vector, the toxicity of the product codedfor by the nucleotide sequence, their secretion characteristics, theirability to fold the polypeptide correctly, their fermentation or culturerequirements, and the ease of purification of the products coded for bythe nucleotide sequence.

The recombinant vector may be an autonomously replicating vector, i.e. avector, which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector is one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the nucleotidesequence encoding the polypeptide of the invention is operably linked toadditional segments required for transcription of the nucleotidesequence. The vector is typically derived from plasmid or viral DNA. Anumber of suitable expression vectors for expression in the host cellsmentioned herein are commercially available or described in theliterature. Useful expression vectors for eukaryotic hosts include, forexample, vectors comprising expression control sequences from SV40,bovine papilloma virus, adenovirus and cytomegalovirus. Specific vectorsare, e.g., pCDNA3.1(+)\Hyg (Invitrogen, Carlsbad, Calif., USA) andpCI-neo (Stratagene, La Jolla, Calif., USA). Useful expression vectorsfor yeast cells include the 2μ plasmid and derivatives thereof, the POT1vector (U.S. Pat. No. 4,931,373), the pJSO37 vector described in Okkels,Ann. New York Acad. Sci. 782, 202-207, 1996, and pPICZ A, B or C(Invitrogen). Useful vectors for insect cells include pVL941, pBG311(Cate et al., “Isolation of the Bovine and Human Genes for MullerianInhibiting Substance And Expression of the Human Gene In Animal Cells”,Cell, 45, pp. 685-98 (1986), pBluebac 4.5 and pMelbac (both availablefrom Invitrogen). Useful expression vectors for bacterial hosts includeknown bacterial plasmids, such as plasmids from E. coli, includingpBR322, pET3a and pET12a (both from Novagen Inc., WI, USA), wider hostrange plasmids, such as RP4, phage DNAs, e.g. the numerous derivativesof phage lambda, e.g. NM989, and other DNA phages, such as M13 andfilamentous single stranded DNA phages.

Other vectors for use in this invention include those that allow thenucleotide sequence encoding the polypeptide to be amplified in copynumber. Such amplifiable vectors are well known in the art. Theyinclude, for example, vectors able to be amplified by DHFR amplification(see, e.g., Kaufman, U.S. Pat. No. 4,470,461, Kaufman and Sharp,“Construction Of A Modular Dihydrafolate Reductase cDNA Gene: AnalysisOf Signals Utilized For Efficient Expression”, Mol. Cell. Biol., 2, pp.1304-19 (1982)) and glutamine synthetase (“GS”) amplification (see,e.g., U.S. Pat. No. 5,122,464 and EP 338,841).

The recombinant vector may further comprise a DNA sequence enabling thevector to replicate in the host cell in question. An example of such asequence (when the host cell is a mammalian cell) is the SV40 origin ofreplication. When the host cell is a yeast cell, suitable sequencesenabling the vector to replicate are the yeast plasmid 2μ replicationgenes REP 1-3 and origin of replication.

The vector may also comprise a selectable marker, e.g. a gene whoseproduct complements a defect in the host cell, such as the gene codingfor dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPIgene (described by P. R. Russell, Gene 40, 1985, pp. 125-130), or onewhich confers resistance to a drug, e.g. ampicillin, kanamycin,tetracyclin, chioramphenicol, neomycin, hygromycin or methotrexate. ForSaccharoinyces cerevisiae, selectable markers include ura3 and leu2. Forfilamentous fungi, selectable markers include amdS, pyrG, arcB, niaD andsC.

The term “control sequences” is defined herein to include all componentswhich are necessary or advantageous for the expression of thepolypeptide of the invention. Each control sequence may be native orforeign to the nucleic acid sequence encoding the polypeptide. Suchcontrol sequences include, but are not limited to, a leader sequence,polyadenylation sequence, propeptide sequence, promoter, enhancer orupstream activating sequence, signal peptide sequence, and transcriptionterminator. At a minimum, the control sequences include a promoter.

A wide variety of expression control sequences may be used in thepresent invention. Such useful expression control sequences include theexpression control sequences associated with structural genes of theforegoing expression vectors as well as any sequence known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof.

Examples of suitable control sequences for directing transcription inmammalian cells include the early and late promoters of SV40 andadenovirus, e.g. the adenovirus 2 major late promoter, the MT-1(metallothionein gene) promoter, the human cytomegalovirusimmediate-early gene promoter (CMV), the human elongation factor 1α(EF-1α) promoter, the Drosophila minimal heat shock protein 70 promoter,the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC)promoter, the human growth hormone terminator, SV40 or adenovirus Elbregion polyadenylation signals and the Kozak consensus sequence (Kozak,M. J Mol Biol 1987 Aug. 20; 196(4):947-50).

In order to improve expression in mammalian cells a synthetic intron maybe inserted in the 5′ untranslated region of the nucleotide sequenceencoding the polypeptide. An example of a synthetic intron is thesynthetic intron from the plasmid pCI-Neo (available from PromegaCorporation, WI, USA).

Examples of suitable control sequences for directing transcription ininsect cells include the polyhedrin promoter, the P10 promoter, theAutographa californiica polyhedrosis virus basic protein promoter, thebaculovirus immediate early gene 1 promoter, the baculovirus 39Kdelayed-early gene promoter, and the SV40 polyadenylation sequence.Examples of suitable control sequences for use in yeast host cellsinclude the promoters of the yeast α-mating system, the yeast triosephosphate isomerase (TPI) promoter, promoters from yeast glycolyticgenes or alcohol dehydrogenase genes, the ADH2-4c promoter, and theinducible GAL promoter. Examples of suitable control sequences for usein filamentous fungal host cells include the ADH3 promoter andterminator, a promoter derived from the genes encoding Aspergillusoryzae TAKA amylase triose phosphate isomerase or alkaline protease, anA. niger α-amylase, A. niger or A. nidulans glucoamylase, A. nidulansacetamidase, Rhiromucor miehei aspartic proteinase or lipase, the TPI1terminator and the ADH3 terminator. Examples of suitable controlsequences for use in bacterial host cells include promoters of the lacsystem, the tip system, the TAC or TRC system, and the major promoterregions of phage lambda.

The presence or absence of a signal peptide will, e.g., depend on theexpression host cell used for the production of the polypeptide to beexpressed (whether it is an intracellular or extracellular polypeptide)and whether it is desirable to obtain secretion. For use in filamentousfungi, the signal peptide may conveniently be derived from a geneencoding an Aspergillus sp. amylase or glucoamylase, a gene encoding aRhizomucor miehei lipase or protease or a Humicola lanuginosa lipase.The signal peptide is preferably derived from a gene encoding A. oryzaeTAKA amylase, A. niger neutral α-amylase, A. niger acid-stable amylase,or A. niger glucoamylase. For use in insect cells, the signal peptidemay conveniently be derived from an insect gene (cf. WO 90/05783), suchas the Lepidopteran manduca sexta adipokinetic hormone precursor, (cf.U.S. Pat. No. 5,023,328), the honeybee melittin (Invitrogen),ecdysteroid UDPglucosyltransferase (egt) (Murphy et al., ProteinExpression and Purification 4, 349-357 (1993) or human pancreatic lipase(hpl) (Methods in Enzymology 284, pp. 262-272, 1997). A preferred signalpeptide for use in mammalian cells is that of hG-CSF or the murine Igkappa light chain signal peptide (Coloma, M (1992) J. Imm Methods152:89-104). For use in yeast cells suitable signal peptides have beenfound to be the α-factor signal peptide from S. cereviciae (cf. U.S.Pat. No. 4,870,008), a modified carboxypeptidase signal peptide (cf. L.A. Valls et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signalpeptide (cf. WO 87/02670), the yeast aspartic protease 3 (YAP3) signalpeptide (cf. M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137), and thesynthetic leader sequence TA57 (WO98/32867). For use in E. coli cells asuitable signal peptide has been found to be the signal peptide ompA.

The nucleotide sequence of the invention encoding a polypeptideexhibiting G-CSF activity, whether prepared by site-directedmutagenesis, synthesis, PCR or other methods, may optionally alsoinclude a nucleotide sequence that encodes a signal peptide. The signalpeptide is present when the polypeptide is to be secreted from the cellsin which it is expressed. Such signal peptide, if present, should be onerecognized by the cell chosen for expression of the polypeptide. Thesignal peptide may be homologous (e.g. be that normally associated withhG-CSF) or heterologous (i.e. originating from another source thanhG-CSF) to the polypeptide or may be homologous or heterologous to thehost cell, i.e. be a signal peptide normally expressed from the hostcell or one which is not normally expressed from the host cell.Accordingly, the signal peptide may be prokaryotic, e.g. derived from abacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian,or insect or yeast cell.

Any suitable host may be used to produce the polypeptide or polypeptidepart of the conjugate of the invention, including bacteria, fungi(including yeasts), plant, insect, mammal, or other appropriate animalcells or cell lines, as well as transgenic animals or plants. Examplesof bacterial host cells include gram-positive bacteria such as strainsof Bacillus, e.g. B. brevis or B. subtilis, or Streptomnyces, orgram-negative bacteria, such as strains of E. coli or Pseudomzonas. Theintroduction of a vector into a bacterial host cell may, for instance,be effected by protoplast transformation (see, e.g., Chang and Cohen,1979, Molecular General Genetics 168: 111-115), using competent cells(see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278). Examples ofsuitable filamentous fungal host cells include strains of Aspergillis,e.g. A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderina.Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspeigillus host cells are described in EP 238 023 andU.S. Pat. No. 5,679,543. Suitable methods for transforming Fusariurnspecies are described by Malardier et al., 1989, Gene 78: 147-156 and WO96/00787. Examples of suitable yeast host cells include strains ofSaccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Klyveromyces,Pichia, such as P. pastoris or P. methanolica, Hansenula, such as H.polymorpha or Yarrowia. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920: and asdisclosed by Clontech Laboratories, Inc, Palo Alto, Calif., USA (in theproduct protocol for the Yeastmaker™ Yeast Transformation System Kit).Examples of suitable insect host cells include a Lepidoptora cell line,such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells(High Five) (U.S. Pat. No. 5,077,214). Transformation of insect cellsand production of heterologous polypeptides therein may be performed asdescribed by Invitrogen. Examples of suitable mammalian host cellsinclude Chinese hamster ovary (CHO) cell lines, (e.g. CHO-K1; ATCCCCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS7 (ATCC CRL-1651)); mouse cells (e.g. NS/O), Baby Hamster Kidney (BHK)cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g.HEK 293 (ATCC CRL-1573)), as well as plant cells in tissue culture.Additional suitable cell lines are known in the art and available frompublic depositories such as the American Type Culture Collection,Rockville, Md. Methods for introducing exogeneous DNA into mammalianhost cells include calcium phosphate-mediated transfection,electroporation, DEAE-dextran mediated transfection, liposome-mediatedtransfection, viral vectors and the transfection method described byLife Technologies Ltd, Paisley, UK using Lipofectamin 2000. Thesemethods are well known in the art and e.g. described by Ausbel et al.(eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons,New York, USA. The cultivation of mammalian cells is conducted accordingto established methods, e.g. as disclosed in (Animal Cell Biotechnology,Methods and Protocols, Edited by Nigel Jenkins, 1999, Human Press Inc,Totowa, N.J., USA and Harrison M A and Rae I F, General Techniques ofCell Culture, Cambridge University Press 1997).

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods known in the art. For example, the cell may becultivated by shake flask cultivation, small-scale or large-scalefermentation (including continuous, batch, fed-batch, or solid statefermentations) in laboratory or industrial fermenters performed in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The resulting polypeptide may be recovered by methods known in the art.For example, the polypeptide may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray drying, evaporation, orprecipitation.

The polypeptides may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989). Specificmethods for purifying polypeptides exhibiting G-CSF activity aredescribed by D. Metcalf and N. A. Nicola in The hemopoieticcolony-stimulating factors, p. 50-51, Cambridge University Press (1995),by C. S. Bae et al, Appl. Microbiol. Biotechnol, 52:338-344 (1999) andin U.S. Pat. No. 4,810,643.

Pharmaceutical Composition of the Invention and its Use

In a further aspect, the present invention comprises a compositioncomprising a polypeptide or conjugate as described herein and at leastone pharmaceutically acceptable carrier or excipient.

The polypeptide, the conjugate or the pharmaceutical compositionaccording to the invention may be used for the manufacture of amedicament for treatment of diseases, in particular prevention ofinfection in cancer patients undergoing certain types of chemotherapy,radiation therapy and bone marrow transplantations, mobilisation ofprogenitor cells for collection in peripheral blood progenitor celltransplantations, treatment of severe chronic or relative leukopenia,treatment of patients with acute myeloid leukaemia, treatment of AIDS orother immunodeficiency diseases, and for antifungal therapy, inparticular for treatment of systemic or invasive candidiasis.

In another aspect the polypeptide, the conjugate or the pharmaceuticalcomposition according to the invention is used in a method of treating amammal having a general haematopoietic disorder, including those arisingfrom radiation therapy or from chemotherapy, in particular neutropeniaor leukopenia, AIDS or other immunodeficiency diseases, comprisingadministering to a mammal in need thereof such a polypeptide, conjugateor pharmaceutical composition. In particular, the method is aimed atincreasing the level of neutrophils in a patient suffering from aninsufficient neutrophil level, for example due to chemotherapy,radiation therapy, or HIV or another viral infection.

The polypeptides and conjugates of the invention will be administered topatients in a “therapeutically effective” dose, i.e. a dose that issufficient to produced the desired effects in relation to the conditionfor which it is administered. The exact dose will depend on the disorderto be treated, and will be ascertainable by one skilled in the art usingknown techniques. The polypeptides or conjugates of the invention maye.g. be administered at a dose similar to that employed in therapy withrhG-CSF such as Neupogen®. A suitable dose of a conjugate of theinvention is contemplated to be in the range of about 5-300 microgram/kgbody weight (based on the weight of the protein part of the conjugate),e.g. 10-200 microgram/kg, such as 25-100 microgram/kg. It will beapparent to those of skill in the art that an effective amount of apolypeptide, conjugate or composition of the invention depends, interalia, upon the disease, the dose, the administration schedule, whetherthe polypeptide or conjugate or composition is administered alone or inconjunction with other therapeutic agents, the serum half-life of thecompositions, the general health of the patient, and the frequency ofadministration. Preferably, the polypeptide, conjugate, preparation orcomposition of the invention is administered in an effective dose, inparticular a dose which is sufficient to normalize the number ofleukocytes, in particular neutrophils, in the patient in question.Normalization of the number of leukocytes may be determined by simplycounting the number of leukocytes at regular intervals in accordancewith established practice.

The polypeptide or conjugate of the invention is preferably administeredin a composition including one or more pharmaceutically acceptablecarriers or excipients. The polypeptide or conjugate can be formulatedinto pharmaceutical compositions in a manner known per se in the art toresult in a polypeptide pharmaceutical that is sufficientlystorage-stable and is suitable for administration to humans or animals.The pharmaceutical composition may be formulated in a variety of forms,including as a liquid or gel, or lyophilized, or any other suitableform. The preferred form will depend upon the particular indicationbeing treated and will be apparent to one of skill in the art.

Accordingly, this invention provides compositions and methods fortreating various forms of leukopenia or neutropenia. In particular thepolypeptide, conjugate or composition of the invention may be used toprevent infection in cancer patients undergoing certain types ofradiation therapy chemotherapy and bone marrow transplantations, tomobilize progenitor cells for collection in peripheral blood progenitorcell transplantations, for treatment of severe chronic or relativeleukopenia and to support treatment of patients with acute myeloidleukaemia. Additionally, the polypeptide, conjugate or composition ofthe invention may be used for treatment of AIDS or otherimmunodeficiency diseases and for antifungal therapy, in particular fortreament of systemic or invasive candidiasis, and for the treatment ofbacterial infections.

Since the polypeptide conjugates of the invention have a long in vivohalf-life and have been found to reduce the duration of neutropenia andleukopenia by administration of a single dose, in contrast to hG-CSFwhich must be administered daily, the conjugates of the invention arewell-suited for administration e.g. on a weekly basis for the preventionand/or treatment of neutropenia. In one embodiment, the polypeptideconjugate or pharmaceutical composition of the invention is for theprevention and/or treatment of neutropenia due to chemotherapy. In thecase of chemotherapy administered at intervals, e.g. on a weekly basisby intravenous injection or by another type of injection, such assubcutaneous or intramuscular injection, it will normally be sufficientto administer the conjugate of the invention in a single dose perchemotherapy treatment, i.e. given either before, after orsimultaneously with the chemotherapy. In other cases where thechemotherapy is administered differently, for example orally on a dailybasis or over an extended period of time by means of an infusion pump,the conjugates of the invention may be administered in a similar manner,e.g. once a week, or, in the case of chemotherapy sessions given lessfrequently than once a week, once per session.

Drug form

The polypeptide or conjugate of the invention can be used “as is” and/orin a salt form thereof. Suitable salts include, but are not limited to,salts with alkali metals or alkaline earth metals, such as sodium,potassium, calcium and magnesium, as well as e.g. zinc salts. Thesesalts or complexes may by present as a crystalline and/or amorphousstructure.

Excipients

“Pharmaceutically acceptable” means a carrier or excipient that at thedosages and concentrations employed does not cause any untoward effectsin the patients to whom it is administered. Such pharmaceuticallyacceptable carriers and excipients are well known in the art (seeRemington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed.,Mack Publishing Company [1990]; Pharmaceutical Formulation Developmentof Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor &Francis [2000]; and Handbook of Pharmaceutical Excipients, 3rd edition,A. Kibbe, Ed., Pharmaceutical Press [2000]).

Mix of Drugs

The pharmaceutical composition of the invention may be administeredalone or in conjunction with other therapeutic agents. These agents maybe incorporated as part of the same pharmaceutical composition or may beadministered separately from the polypeptide or conjugate of theinvention, either concurrently or in accordance with another treatmentschedule. In addition, the polypeptide, conjugate or pharmaceuticalcomposition of the invention may be used as an adjuvant to othertherapies.

Patients

A “patient” for the purposes of the present invention includes bothhumans and other mammals. Thus the methods are applicable to both humantherapy and veterinary applications.

Administration Route

The administration of the formulations of the present invention can beperformed in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraocularly, or in any other acceptable manner.The formulations can be administered continuously by infusion, althoughbolus injection is acceptable, using techniques well known in the art.Typically, the formulation will designed for parenteral administration,e.g. by the subcutaneous route.

Parenterals

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations may also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by thoseskilled in the art that lyophilized preparations are generally morestable than their liquid counterparts. Such lyophilized preparations arereconstituted prior to use by the addition of one or more suitablepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution.

In case of parenterals, they are prepared for storage as lyophilizedformulations or aqueous solutions by mixing, as appropriate, thepolypeptide having the desired degree of purity with one or morepharmaceutically acceptable carriers, excipients or stabilizerstypically employed in the art (all of which are termed “excipients”),for example buffering agents, stabilizing agents, preservatives,isotonifiers, non-ionic detergents, antioxidants and/or othermiscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from about 2 mM to about 50 mM Suitable buffering agents for usewith the present invention include both organic and inorganic acids andsalts thereof such as citrate buffers (e.g., monosodium citrate-disodiumcitrate mixture, citric acid-trisodium citrate mixture, citricacid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typicallyadded in amounts of about 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides (e.g. benzalkonium chloride, bromide oriodide), hexamethonium chloride, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, omithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran.Stabilizers are typically present in the range of from 0.1 to 10,000parts by weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe present to help solubilize the therapeutic agent as well as toprotect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.).

Additional miscellaneous excipients include bulking agents or fillers(e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g.,ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

Parenteral formulations to be used for in vivo administration must besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Sustained Release Preparations

Suitable examples of sustained-release preparations includesemi-permeable s5 matrices of solid hydrophobic polymers containing thepolypeptide or conjugate, the matrices having a suitable form such as afilm or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot® (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S-S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

All references cited herein are hereby incorporated by reference intheir entirety for all purposes.

The invention is further described in the non-limiting examples below.

DESCRIPTION OF THE DRAWINGS

FIG. 1: The in vivo half-lives of rhG-CSF (Neupogen®) and SPA-PEG5000-conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K

FIG. 2: The in vivo half-lives of rhG-CSF (Neupogen®) and SPA-PEG5000-conjugated hG-CSF K16R K34R K40R Q90K T105K Q159K

FIG. 3: The in vivo biological activities in healthy rats of rhG-CSF(Neupogen®), SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70K Q120Kand SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K.

FIG. 4: The in vivo biological activities in healthy rats of rhG-CSF(Neupogen®), SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70K Q120KT133K and SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q90K Q120KT133K.

FIG. 5: The in vivo biological activities in healthy rats of rhG-CSF(Neupogen®), SPA-PEG 12000-conjugated hG-CSF K16R K34R K40R anddifferent doses of SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70KQ90K Q120K.

FIG. 6: The in vivo biological activities in healthy rats of rhG-CSF(Neupogen®), SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q70K Q90KQ120K, SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R Q90K T105K S159Kand SPA-PEG 20000-conjugated hG-CSF K16R K34R K40R T105K S159K.

FIG. 7: The in vivo biological activities in rats withchemotherapy-induced neutropenia of rhG-CSF (Neupogen®), SPA-PEG5000-conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K, SPA-PEG20000-conjugated hG-CSF K16R K34R K40R Q90K.

FIG. 8: The in vivo biological activities (white blood cell count) inrats with chemotherapy-induced neutropenia of rhG-CSF (Neupogen®),SPA-PEG 5000-conjugated hG-CSF K16R K34R K40R T105K S159K, and SPA-PEG5000-conjugated hG-CSF K16R K34R K40R Q90K T105K S159K.

FIG. 9: The in vivo biological activities (absolute neutrophil count) inrats with chemotherapy-induced neutropenia of rhG-CSF (Neupogen®) andSPA-PEG 5000-conjugated hG-CSF K16R K34R K40R T105K S159K.

FIG. 10: The in vivo biological activities (white blood cell count) inrats with chemotherapy-induced neutropenia of rhG-CSF (Neupogen®),rhG-CSF with a 20 kDa N-terminal PEG group (Neulasta™), and SPA-PEG5000-conjugated hG-CSF K16R K34R K40R T105K S159K produced in yeast andin CHO cells.

FIG. 11: The in vivo biological activities (absolute neutrophil count)in rats with chemotherapy-induced neutropenia of rhG-CSF (Neupogen®),rhG-CSF with a 20 kDa N-terminal PEG group (Neulasta™), and SPA-PEG5000-conjugated hG-CSF K16R K34R K40R T105K S159K produced in yeast andin CHO cells.

SEQUENCE LISTING

The appended sequence listing contains the following sequences:

-   SEQ ID NO:1: The amino acid sequence of human G-CSF.-   SEQ ID NO:2: A synthetic DNA sequence encoding human G-CSF, with    codon usage optimized for expression in E. coli.-   SEQ ID NO:3: The amino acid sequence of the OmpA signal sequence.-   SEQ ID NO:4: A synthetic DNA sequence encoding the OmpA signal    sequence.-   SEQ ID NO:5: A synthetic histidine tag.-   SEQ ID NO:6: A synthetic DNA sequence encoding the histidine tag of    SEQ ID NO:5.-   SEQ ID NO:7: The amino acid sequence of a human G-CSF signal    peptide.-   SEQ ID NO:8: A synthetic DNA sequence encoding human G-CSF,    including the signal peptide of SEQ ID NO:7, with codon usage    optimized for expression in CHO cells.-   SEQ ID NO:9-15: Various synthetic tags    Materials and Methods    Methods Used to Determine the Amino Acids to be Modified    Accessible Surface Area (ASA)

A 3D ensemble of 10 structures determined by NMR spectroscopy (Zink etal. (1994) Biochemistry 33: 8453-8463) is available from the ProteinData Bank (PDB) (www.rcsb.org/pdb/). This information can be enteredinto the computer program Access (B. Lee and F. M. Richards, J. Mol.Biol. 55: 379-400 (1971)) version 2 (© 1983 Yale University) and used tocompute the accessible surface area (ASA) of the individual atoms in thestructure. This method typically uses a probe size of 1.4Å and definesthe Accessible Surface Area (ASA) as the area formed by the centre ofthe probe. Prior to this calculation all water molecules and allhydrogen atoms should be removed from the coordinate set as should otheratoms not directly related to the protein.

Fractional ASA of Side Chain

The fractional ASA of the side chain atoms is computed by division ofthe sum of the ASA of the atoms in the side chain with a valuerepresenting the ASA of the side chain atoms of that residue type in anextended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991)J. Mol. Biol. 220, 507-530. For this example the CA atom is regarded asa part of the side chain of glycine residues but not for the remainingresidues. The values in the following table are used as standard 100%ASA for the side chain:

Ala 69.23 Å² Arg 200.35 Å² Asn 106.25 Å² Asp 102.06 Å² Cys 96.69 Å² Gln140.58 Å² Glu 134.61 Å² Gly 32.28 Å² His 147.00 Å² Ile 137.91 Å² Leu140.76 Å² Lys 162.50 Å² Met 156.08 Å² Phe 163.90 Å² Pro 119.65 Å² Ser78.16 Å² Thr 101.67 Å² Trp 210.89 Å² Tyr 176.61 Å² Val 114.14 Å²

Residues not detected in the structure are defined as having 100%exposure as they are thought to reside in flexible regions.

Determining Distances Between Atoms:

The distance between atoms is most easily determined using moleculargraphics software, e.g. InsightII® v. 98.0, MSI INC.

General Considerations Regarding Amino Acid Residues to be Modified

As explained above, amino acid residues to be modified in accordancewith the present invention are preferably those whose side chains aresurface exposed, in particular those with more than about 25% of theside chain exposed at the surface of the molecule, and more preferablythose with more than 50% side chain exposure. Another consideration isthat residues located in receptor interfaces are preferably excluded soas to avoid or at least minimize possible interference with receptorbinding or activation. A further consideration is that residues that areless than 10 Å from the nearest Lys (Glu, Asp) CB-CB (CA for Gly) shouldalso be excluded. Finally, preferred positions for modification are inparticular those that have a hydrophilic and/or charged residue, i.e.Asp, Asn, Glu, Gln, Arg, His, Tyr, Ser and Thr, positions that have anarginine residue being especially preferred.

Identifying G-CSF Amino Acid Residues for Modification

The information below illustrates the factors that generally should betaken into consideration when identifying amino acid residues to bemodified in accordance with the present invention.

Three-dimensional structures have been reported for human G-CSF by X-raycrystallography (Hill et al. (1993) Proc. Natl. Acad. Sci. USA 90:5167-5171) and by NMR spectroscopy (Zink et al. (1994) Biochemistry 33:8453-8463). As mentioned above, Aritomi et al. (Nature 401:713-717,1999) have identified the following hG-CSF residues as being part of thereceptor binding interfaces: G4, P5, A6, S7, S8, L9, P10, Q11, S12, L15,K16, E19, Q20, L108, D109, D112, T115, T116, Q119, E122, E123, and L124.Thus, although it is possible to modify these residues, it is preferredthat these residues are excluded from modification.

Using the 10 NMR structures of G-CSF identified by Zink et al. (1994) asinput structures followed by a computation of the average ASA of theside chain, the following residues have been identified as having morethan 25% ASA: M0, T1, P2, L3, G4, P5, A6, S7, S8, L9, P10, Q11, S12,F13, L14, L15, K16, C17, E19, Q20, V21, R22, K23, Q25, G26, D27, A29,A30, E33, K34, C36, A37, T38, Y39, K40, L41, H43, P44, E45, E46, V48,L49, L50, H52, S53, L54, I56, P57, P60, L61, S62, S63, P65, S66, Q67,A68, L69, Q70, L71, A72, G73, C74, S76, Q77, L78, S80, F83, Q86, G87,Q90, E93, G94, S96, P97, E98, L99, G100, P101, T102, D104, T105, Q107,L108, D109, A111, D112, F113, T115, T116, W118, Q119, Q120, M121, E122,E123, L124, M126, A127, P128, A129, L130, Q131, P132, T133, Q134, G135,A136, M137, P138, A139, A141, S142, A143, F144, Q145, R146, R147, S155,H156, Q158, S159, L161, E162, V163, S164, Y165, R166, V167, L168, R169,H170, L171, A172, Q173, P174.

Similarly, the following residues have more than 50% ASA: M0, T1, P2,L3, G4, P5, A6, S7, S8, L9, P10, Q11, S12, F13, L14, L15, K16, C17, E19,Q20, R22, K23, G26, D27, A30, E33, K34, T38, K40, L41, H43, P44, E45,E46, L49, L50, S53, P57, P60, L61, S62, S63, P65, S66, Q67, A68, L69,Q70, L71, A72, G73, S80, F83, Q90, G94, P97, E98, P101, D104, T105,L108, D112, F113, T115, T116, Q119, Q120, E122, E123, L124, M126, P128,A129, L130, Q131, P132, T133, Q134, G135, A136, A139, A141, S142, A143,F144, R147, S155, S159, E162, R166, V167, R169, H170, L171, A172, Q173,P174.

The molecular graphics program InsightII® v.98.0 was used to determineresidues having their CB atom (CA in the case of glycine) at a distanceof more than 15 Å from the nearest amine group, defined as the NZ atomsof lysine and the N atom of the N-terminal residue T1. The followinglist includes the residues that fulfill this criteria in at least one ofthe 10 NMR structures. G4, P5, A6, S7, S8, L9, P10, Q11, L14, L15, L18,V21, R22, Q25, G26, D27, G28, A29, Q32, L35, C36, T38, Y39, C42, H43,P44, E45, E46, L47, V48, L49, L50, G51, H52, S53, L54, G55, I56, P57,W58, A59, P60, L61, S62, S63, C64, P65, S66, Q67, A68, L69, Q70, L71,A72, G73, C74, L75, S76, Q77, L78, H79, S80, G81, L82, F83, L84, Y85,Q86, G87, L88, L89, Q90, A91, L92, E93, G94, I95, S96, P97, E98, L99,G100, P101, T102, L103, D104, T105, L106, Q107, L108, D109, V110, A111,D112, F113, A114, T115, T116, I117, W118, Q119, Q120, M121, E122, E123,L124, G125, M126, A127, P128, A129, L130, Q131, P132, T133, Q134, G135,A136, M137, P138, A139, F140, A141, S142, A143, F144, Q145, R146, R147,A148, G149, G150, V151, L152, V153, A154, S155, H156, L157, Q158, S159,F160, L161, E162, V163, S164, Y165, R166, V167, L168, R169, H170, L171,A172, Q173, P174.

The InsightII® v.98.0 program was similarly used to determine residueshaving their CB atom (CA atom in the case of glycine) at a distance ofmore than 10 Å from the nearest acidic group, defined as the CG atoms ofaspartic acid, the CD atoms of glutamic acid and the C atom of theC-terminal residue P174. The following list includes the residues thatfulfill this criteria in at least one of the 10 NMR structures. M0, T1,P2, L3, G4, P5, A6, S7, S8, L9, P10, Q11, S12, F13, L14, T38, Y39, K40,L41, C42, L50, G51, H52, S53, L54, G55, I56, P57, W58, A59, P60, L61,S62, S63, C64, P65, S66, Q67, A68, L69, Q70, L71, A72, G73, C74, L75,S76, Q77, L78, H79, S80, G81, L82, F83, L84, Y85, Q86, G87, L88, I117,M126, A127, P128, A129, L130, Q131, P132, T133, Q134, G135, A136, M137,P138, A139, F140, A141, S142, A143, F144, Q145, R146, R147, A148, G149,G150, V151, L152, V153, A154, S155, H156, L157, V167, L168, R169, H170,L171.

By combining and comparing the above lists, it is possible to selectindividual amino acid residues for modification to result in a listcontaining a limited number of amino acid residues whose modification ina given G-CSF polypeptide is likely to result in desired properties.

Methods for PEGylation of hG-CSF and Variants Thereof

PEGylation of hG-CSF and Variants Thereof in Solution

Human G-CSF and variants thereof are PEGylated at a concentration of 250μg/ml in 50 mM sodium phosphate, 100 mM NaCl, pH 8.5. The molar surplusof PEG is 50-100 times with respect to PEGylation sites on the protein.The reaction mixture is placed in a thermo mixer for 30 minutes at 37°C. at 1200 rpm. After 30 minutes, quenching of the reaction is obtainedby adding a molar excess of glycine.

Cation exchange chromatography is applied to remove excess PEG, glycineand other by-products from the reaction mixture. The PEGylation reactionmixture is diluted with 20 mM sodium citrate pH 2.5 until the ionicstrength is less than 7 mS/cm. pH is adjusted to 2.5 using 5 N HCl. Themixture is applied to a SP-sepharose FF column equilibrated with 20 mMsodium citrate pH 2.5. Unbound material is washed off the column using 4column volumes of equilibration buffer. PEGylated protein is eluted inthree column volumes by adding 20 mM sodium citrate, 750 mM sodiumchloride. Pure PEGylated G-CSF is concentrated and buffer exchange isperformed using VivaSpin concentration devices, molecular weight cut-off(mwco): 10 kDa.

PEGylation in Microtiter Plates of a Tagged Polypeptide with G-CSFActivity

A polypeptide exhibiting G-CSF activity is expressed with a suitabletag, e.g. any of the tags exemplified in the general description above,and culture broth is transferred to one or more wells of a microtiterplate capable of immobilising the tagged polypeptide. When the tag isMet-Lys-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-His-Gln-Gln (SEQ IDNO:5), a nickel-nitrilotriacetic acid (Ni-NTA) HisSorb microtiter platecommercially available from QIAGEN can be used.

After immobilization of the tagged polypeptide to the microtiter plate,the wells are washed in a buffer suitable for binding and subsequentPEGylation followed by incubating the wells with the activated PEG ofchoice. As an example, M-SPA-5000 from Shearwater Corp. is used. Themolar ratio of activated PEG to polypeptide should be optimized, butwill typically be greater than 10:1, e.g. up to about 100:1 or higher.After a suitable reaction time at ambient temperature, typically around1 hour, the reaction is stopped by removal of the activated PEGsolution. The conjugated protein is eluted from the plate by incubationwith a suitable buffer. Suitable elution buffers may contain imidazole,excess NTA or another chelating compound. The conjugated protein isassayed for biological activity and immunogenicity as appropriate. Thetag may optionally be cleaved off using a method known in the art, e.g.using diaminopeptidase the Gln in pos-1 can be converted to pyroglutamylwith GCT (glutamylcyclotransferase) and finally cleaved off with PGAP(pyro-glutamyl-aminopeptidase), giving the untagged protein. The processinvolves several steps of metal chelate affinity chromatography.Alternatively, the tagged polypeptide may be conjugated.

PEGylation of a Polypeptide Exhibiting hG-CSF Activity and having aBlocked Receptor-Binding Site

In order to optimize PEGylation of hG-CSF in a manner excludingPEGylation of lysines involved in receptor recognition, the followingmethod has been developed:

Purified hG-CSF is obtained as described in Example 4. A homodimercomplex consisting of an hG-CSF polypeptide and the soluble domain ofthe G-CSF receptor in a 2:2 stochiometry is formed in aphosphate-buffered saline solution (PBS) buffer at pH 7. Theconcentration of hG-CSF polypeptide is approximately 20 μg/ml or 1 μMand the receptor is present at an equimolar concentration.

M-SPA-5000 from Shearwater Corp. is added at 3 different concentrationlevels corresponding to a 10, 25 and 50 molar excess to the number ofattachment sites in hG-CSF polypeptide. The reaction time is 30 min atroom temperature. After the 30 min reaction period, the pH of thereaction mixture is adjusted to pH 2.0 and the reaction mixture isapplied to a Vydac C18 column and eluted with an acetonitrile gradientessentially as described (Utsumi et al., J. Biochem., Vol. 101,1199-1208, (1987). Alternatively, an isopropanol gradient can be used.

Fractions are analyzed using the primary screening assay describedherein and active PEGylated hG-CSF polypeptide obtained by this methodis stored at −80° C. in PBS, pH 7 containing 1 mg/ml human serum albumin(HSA).

Methods Used to Characterize Conjugated and Non-Conjugated hG-CSF andVariants Thereof.

Determination of the Molecular Size of hG-CSF and Variants Thereof.

The molecular weight of conjugated or non-conjugated hG-CSF or variantsthereof is determined by either SDS-PAGE, gel filtration, matrixassisted laser desorption mass spectrometry or equilibriumcentrifugation. As explained above, SDS-PAGE provides information on the“apparent molecular weight”. The actual molecular weight canadvantageously be determined using mass spectrometry. SDS-PAGE iscarried out using the NuPAGE® kit (Novex high-performance pre-cast gels)from Invitrogen™. 15 μl of the samples are loaded onto NuPAGE 4-12%Bis-Tris gels (Cat. Nr. NPO321) and eluted in NuPAGE MES SDS runningbuffer (Cat. Nr. NPO002-02) for 35 minutes at 200 V and 120 mA.

Determination of Polypeptide Concentration

The concentration of a polypeptide can be measured using optical densitymeasurements at 280 nm, an enzyme-linked immunoadsorption assay (ELISA),a radio-immunoassay (RIA), or other such immunodetection techniques wellknown in the art. Furthermore, the polypeptide concentration in a samplecan be measured with the Biacore® instrument using a Biacore® chipcoated with an antibody specific for the polypeptide.

Such an antibody can be coupled covalently to the Biacore® chip byvarious chemistries. Alternatively, the antibody can be boundnon-covalently e.g. by means of an antibody specific for the Fc portionof the anti-polypeptide antibody. The Fc specific antibody is firstcoupled covalently to the chip. The anti-polypeptide antibody is thenflowed over the chip and is bound by the first antibody in a directedfashion. Furthermore, biotinylated antibodies can be immobilized using astreptavidin coated surface (e.g. Biacore Sensor Chip SA®) (Real-TimeAnalysis of Biomolecular Interactions, Nagata and Handa (Eds.), 2000,Springer Verlag, Tokyo; Biacore 2000 Instrument Handbook, 1999, BiacoreAB).

When the sample is flowed over the chip the polypeptide will bind to thecoated antibody and the increase in mass can be measured. By using apreparation of the polypeptide in a known concentration, a standardcurve can be established and subsequently the concentration of thepolypeptide in the sample can be determined. After each injection ofsample the sensor chip is regenerated by a suitable eluent (e.g. a lowpH buffer) that removes the bound analyte.

Generally, the applied antibodies will be monoclonal antibodies raisedagainst the wild type polypeptide. Introduction of mutations or othermanipulations of the wild type polypeptide (extra glycosylations orpolymer conjugations) may alter the recognition by such antibodies.Furthermore, such manipulations that give rise to an increased molecularweight of the polypeptide will result in an increased plasmon resonancesignal. Consequently, it is necessary to establish a standard curve forevery molecule to be tested.

Methods Used to Determine the In Vitro and In Vivo Activity ofConjugated and Non-Conjugated hG-CSF and Variants Thereof.

Primary Assay 1—In Vitro G-CSF Activity Assay

Proliferation of the murine cell line NFS-60 (obtained from Dr. J. me,St. Jude Children's Research Hospital, Tennessee, USA) is dependent onthe presence of active G-SCF in the growth medium. Thus, the in vitrobiological activity of hG-CSF and variants thereof can be determined bymeasuring the number of dividing NFS-60 cells after addition of a G-CSFsample to the growth medium followed by incubation over a fixed periodof time.

NFS-60 cells are maintained in Iscoves DME Medium containing 10% w/w FBS(fetal bovine serum), 1% w/w Pen/Strep, 10 μg per litre hG-CSF and 2 mMGlutamax. Prior to sample addition, cells are washed twice in growthmedium without hG-CSF and diluted to a concentration of 2.2×10⁵ cellsper ml. 100 μl of the cell suspension is added to each well of a 96 wellmicrotiter plate (Corning).

Samples containing conjugated or non-conjugated G-CSF or variantsthereof are diluted to concentrations between 1.1×10⁻⁶ M and 1.1×10⁻ ¹³M in the growth medium. 10 μl of each sample is added to 3 wellscontaining NFS-60 cells. A control consisting of 10 μl of mammaliangrowth medium is added to 8 wells on each microtiter plate. The cellsare incubated for 48 hours (37° C., 5% CO₂) and the number of dividingcells in each well is quantified using the WST-1 cell proliferationagent (Roche Diagnostics GmbH, Mannheim, Germany). 0.01 ml WST-1 isadded to the wells followed by incubation for 150 min. at 37° C. in a 5%CO₂ air atmosphere. The cleavage of the tetrazolium salt WST-1 bymitochondrial dehydrogenases in viable cells results in the formation offormazan that is quantified by measuring the absorbance at 450 nm.Hereby, the number of viable cells in each well is quantified.

Based on these measurements, dose-response curves for each conjugatedand non-conjugated G-CSF molecule or variants thereof are calculated,after which the EC50 value for each molecule can be determined. Thisvalue is equal to the amount of active G-CSF protein that is necessaryto obtain 50% of the maximum proliferation activity of non-conjugatedhuman G-CSF. Thus, the EC50 value is a direct measurement of the invitro activity of the given protein, a lower EC50 value indicating ahigher activity.

Primary Assay 2—In Vitro G-CSF Activity Assay

The murine hematopoietic cell line BaF3 is transfected with a plasmidcarrying the human G-CSF receptor and the promoter of the transcriptionregulator, fos, in front of the luciferase reporter gene. Uponstimulation of such a cell line with a G-CSF sample, a number ofintracellular reactions lead to stimulation of fos expression, andconsequently to expression of luciferase. This stimulation is monitoredby the Steady-Glo™ Luciferase Assay System (Promega, Cat. No. E2510)whereby the in vitro activity of the G-CSF sample may be quantified.

BaF3/hGCSF-R/pfos-lux cells are maintained at 37° C. in a humidified 5%CO₂ atmosphere in complete culture media (RPMI-1640/HEPES (Gibco/BRL,Cat. No. 22400), 10% FBS (HyClone, characterized), 1×Penicillin/Streptomycin (Gibco/BRL, Cat. No. 15140-122), 1× L-Glutamine(Gibco/BRL, Cat. No. 25030-081), 10% WEHI-3 conditioned media (source ofmuIL-3), and grown to a density of 5×10⁵ cells/mL (confluent). The cellsare reseeded at about 2×10⁴ cells/mL every 2-3 days.

One day prior to the assay, log-phase cells are resuspended at 2×10⁵cells/mL in starving media (DMEMIF-12 (Gibco/BRL, Cat. No. 11039), 1%BSA (Sigma, Cat. No. A3675), 1× Penicillin/Streptomycin (Gibco/BRL, Cat.No. 15140-122), 1× L-Glutamine (Gibco/BRL, Cat. No. 25030-081), 0.1%WEHI-3 conditioned media) and starved for 20 hours. The cells are washedtwice with PBS and tested for viability using Trypan Blue viabilitystaining. The cells are resuspended in assay media (RPMI-1640(phenol-red free, Gibco/BRL, Cat. No. 11835), 25 mM HEPES, 1% BSA(Sigma, Cat. No. A3675), 1× Penicillin/Streptomycin (Gibco/BRL, Cat. No.15140-122), 1× L-Glutamine (Gibco/BRL, Cat. No. 25030-081) at 4×10⁶cells/mL, and 50 μL are aliquotted into each well of a 96-wellmicrotiter plate (Corning). Samples containing conjugated ornon-conjugated G-CSF or variants thereof are diluted to concentrationsbetween 1.1×10⁻⁷ M and 1.1×10⁻¹² M in the assay medium. 50 μl of eachsample is added to 3 wells containing BaF3/hGCSF-R/pfos-lux cells. Anegative control consisting of 50 μl of medium is added to 8 wells oneach microtiter plate. The plates are mixed gently and incubated for 2hours at 37° C. The luciferase activity is measured by following thePromega Steady-Glo™ protocol (Promega Steady-Glo™ Luciferase AssaySystem, Cat. No. E2510). 100 μL of substrate is added per well followedby gentle mixing. Luminescence is measured on a TopCount luminometer(Packard) in SPC (single photon counting) mode.

Based on these measurements, dose-response curves for each conjugatedand non-conjugated G-CSF molecule or variants thereof are calculated,after which the EC50 value for each molecule can be determined.

Secondary Assay—Binding Affinity of G-CSF or Variants Thereof to thehG-CSF Receptor

Binding of rhG-CSF or variants thereof to the hG-CSF receptor is studiedusing standard binding assays. The receptors may be purifiedextracellular receptor domains, receptors bound to purified cellularplasma membranes, or whole cells—the cellular sources being either celllines that inherently express G-CSF receptors (e.g. NFS-60) or cellstransfected with cDNAs encoding the receptors. The ability of rhG-CSF orvariants thereof to compete for the binding sites with native G-CSF isanalyzed by incubating with a labeled G-CSF-analog, for instancebiotinylated hG-CSF or radioiodinated hG-CSF. An example of such anassay is described by Yamasaki et al. (Drugs. Exptl. Clin. Res.24:191-196 (1998)).

The extracellular domains of the hG-CSF receptor can optionally becoupled to Fc and immobilized in 96 well plates. RhG-CSF or variantsthereof are subsequently added and the binding of these is detectedusing either specific anti-hG-CSF antibodies or biotinylated orradioiodinated hG-CSF.

Measurement of the In Vivo Half-Life of Conjugated and Non-ConjugatedrhG-CSF and Variants Thereof.

An important aspect of the invention is the prolonged biologicalhalf-life that is obtained by construction of a hG-CSF with or withoutconjugation of the polypeptide to the polymer moiety. The rapid decreaseof hG-CSF serum concentrations has made it important to evaluatebiological responses to treatment with conjugated and non-conjugatedhG-CSF and variants thereof. Preferably, the conjugated andnon-conjugated hG-CSF and variants thereof of the present invention haveprolonged serum half-lives also after i.v. administration, making itpossible to measure by e.g. an ELISA method or by the primary screeningassay. Measurement of in vivo biological half-life was carried out asdescribed below.

Male Sprague Dawley rats (7 weeks old) were used. On the day ofadministration, the weights of the animals were measured (280-310 gramper animal). 100 μg per kg body weight of the non-conjugated andconjugated hG-CSF samples were each injected intravenously into the tailvein of three rats. At 1 minute, 30 minutes, 1, 2, 4, 6, and 24 hoursafter the injection, 500 μl of blood was withdrawn from the eyes of eachrat while under CO₂-anaesthesia. The blood samples were stored at roomtemperature for 1 ½ hours followed by isolation of serum bycentrifugation (4° C., 18000×g for 5 minutes). The serum samples werestored at −80° C. until the day of analysis. The amount of active G-CSFin the serum samples was quantified by the G-CSF in vitro activity assay(see primary assay 2) after thawing the samples on ice.

Another example of an assay for the measurement of in vivo half-life ofG-CSF or variants thereof is described in U.S. Pat. No. 5,824,778, thecontent of which is hereby incorporated by reference.

Measurement of the In Vivo Biological Activity in Healthy Rats ofConjugated and Non-Conjugated hG-CSF and Variants Thereof.

Measurement of the in vivo biological effects of hG-CSF in SPF SpragueDawley rats (purchased from M & B A/S, Denmark) is used to evaluate thebiological efficacy of conjugated and non-conjugated G-CSF and variantsthereof.

On the day of arrival the rats are randomly allocated into groups of 6.The animals are acclimatized for a period of 7 days wherein individualsin poor condition or at extreme weights are rejected. The weight rangeof the rats at the start of the acclimatization period is 250-270 g.

On the day of administration the rats are fasted for 16 hours followedby subcutaneous injection of 100 μg per kg body weight of hG-CSF or avariant thereof. Each hG-CSF sample is injected into a group of 6randomized rats. Blood samples of 300 μl EDTA stabilized blood are drawnfrom a tail vein of the rats prior to dosing and at 6, 12, 24, 36, 48,72, 96, 120 and 144 hours after dosing. The blood samples are analyzedfor the following haematological parameters: Haemoglobin, red blood cellcount, haematocrit, mean cell volume, mean cell haemoglobinconcentration, mean cell haemoglobin, white blood cell count,differential leucocyte count (neutrophils, lymphocytes, eosinophils,basophils, monocytes). On the basis of these measurements the biologicalefficacy of conjugated and non-conjugated hG-CSF and variants thereof isevaluated. Further examples of assays for the measurement of in vivobiological activity of hG-CSF or variants thereof are described in U.S.Pat. No. 5,681,720, U.S. Pat. No. 5,795,968, U.S. Pat. No. 5,824,778,U.S. Pat. No. 5,985,265 and by Bowen et al., Experimental Hematology27:425-432 (1999).

Measurement of the In Vivo Biological Activity in Rats withChemotherapy-Induced Neutropenia of Conjugated and Non-Conjugated hG-CSFand Variants Thereof

SPF Sprague Dawley rats were purchased from M & B A/S, Denmark. On theday of arrival the rats are randomly allocated into groups of 6. Theanimals are acclimatized for a period of 7 days wherein individuals inpoor condition or at extreme weights are rejected. The weight range ofthe rats at the start of the acclimatization period is 250-270 g.

24 hours before administration of the hG-CSF samples the rats areinjected i.p. with 50 or 90 mg per kg body weight of cyclophosphamide(CPA). The PEGylated hG-CSF variants are given as a single dose injecteds.c. at day 0, while non-conjugated hG-CSF is injected s.c. either in asingle dose at day 0 or on a daily basis. For hG-CSF or variants givenin a single dose at day 0, the dosage is 100 μg per kg body weight. Fornon-conjugated hG-CSF (Neupogen®) given on a daily basis, the dosagevaried and is given in the examples below. Each hG-CSF sample isinjected into a group of 6 randomized rats. Blood samples of 300 μl EDTAstabilized blood are drawn from a tail vein of the rats prior to dosingand at 6, 12, 24, 36, 48, 72, 96, 120, 144 and 168 hours after dosing.The blood samples are analyzed for the following haematologicalparameters: hemoglobin, red blood cell count, haematocrit, mean cellvolume, mean cell haemoglobin concentration, mean cell haemoglobin,white blood cell count, differential leucocyte count (neutrophils,lymphocytes, eosinophils, basophils, monocytes). On the basis of thesemeasurements the biological efficacy of conjugated and non-conjugatedhG-CSF and variants thereof is evaluated.

Determination of Polypeptide Receptor-Binding Affinity (On- andOff-Rate)

The strength of the binding between a receptor and ligand can bemeasured using an enzyme-linked immunoadsorption assay (ELISA), aradio-immunoassay (RIA), or other such immunodetection techniques wellknown in the art. The ligand-receptor binding interaction may also bemeasured with the Biacore® instrument, which exploits plasmon resonancedetection (Zhou et al., Biochemistry, 1993, 32, 8193-98; Faegerstram andO'Shannessy, 1993, In Handbook of Affinity Chromatography, 229-52,Marcel Dekker, Inc., NY).

The Biacore® technology allows one to bind receptor to a gold surfaceand to flow ligand over it. Plasmon resonance detection gives directquantification of the amount of mass bound to the surface in real time.This technique yields both on- and off-rate constants and thus aligand-receptor dissociation constant and an affinity constant can bedirectly determined.

In Vitro Immunogenicity Test of hG-CSF Conjugates

The reduced immunogenicity of a conjugate of the invention can bedetermined by use of an ELISA method measuring the immunoreactivity ofthe conjugate relative to a reference molecule or preparation. Thereference molecule or preparation is normally a recombinant human G-CSFpreparation such as Neupogen® or another recombinant human G-CSFpreparation, e.g. an N-terminally PEGylated rhG-CSF molecule asdescribed in U.S. Pat. No. 5,824,784. The ELISA method is based onantibodies from patients treated with one of these recombinant G-CSFpreparations. The immunogenicity is considered to be reduced when theconjugate of the invention has a statistically significant lowerresponse in the assay than the reference molecule or preparation.

Neutralisation of activity in G-CSF Bioassay

The neutralisation of hG-CSF conjugates by anti-G-CSF sera is analyzedusing the G-CSF bioassay described above.

Sera from patients treated with the G-CSF reference molecule or fromimmunized animals are used. Sera are added either in a fixedconcentration (dilution 1:20-1:500 (pt sera) or 20-1000 ng/ml (animalsera)) or in five-fold serial dilutions of sera starting at 1:20 (ptsera) or 1000 ng/ml (animal sera). HG-CSF conjugate is added either inseven fold-dilutions starting at 10 nM or in a fixed concentration(1-100 pM) in a total volume of 80 μl DMEM medium +10% FCS. The sera areincubated for 1 hr. at 37° C. with hG-CSF conjugate.

The samples (0.01 ml) are then transferred to 96 well tissue cultureplates containing NFS-60 cells in 0.1 ml DMEM media. The cultures areincubated for 48 hours at 37° C. in a 5% CO₂ air atmosphere. 0.01 mlWST-1 (WST-1 cell proliferation agent, Roche Diagnostics GmbH, Mannheim,Germany) is added to the cultures and incubated for 150 min. at 37° C.in a 5% CO₂ air atmosphere. The cleavage of the tetrazolium salt WST-1by mitochondrial dehydrogenases in viable cells results in the formationof formazan that is quantified by measuring the absorbance at 450 nm.

When hG-CSF conjugate samples are titrated in the presence of a fixedamount of serum, the neutralising effect is defined as fold inhibition(FI) quantified as EC50 (with serum)/EC50 (without serum). The reductionof antibody neutralisation of G-CSF variant proteins is defined as

$\left( {1 - \frac{\left( {{{FI}\mspace{20mu}{variant}} - 1} \right)}{\left( {{{FI}\mspace{20mu}{wt}} - 1} \right)}} \right) \times 100\%$

EXAMPLE 1

Construction and Cloning of Synthetic Genes Encoding hG-CSF

The following DNA fragments were synthesized following the generalprocedure described by Stemmer et al. (1995), Gene 164, pp. 49-53:

Fragment 1, consisting of a Bam HI digestion site, a sequence encodingthe YAP3 signal peptide (WO 98/32867), a sequence encoding the TA57leader sequence (WO 98/32867), a sequence encoding a KEX2 proteaserecognition site (AAAAGA), a sequence encoding hG-CSF with its codonusage optimized for expression in E. coli, (SEQ ID NO:2) and a Xba Idigestion site.

Fragment 2, consisting of a Bam HI digestion site, a sequence encodingthe YAP3 signal peptide (WO 98/32867), a sequence encoding the TA57leader sequence (WO 98/32867), a sequence encoding a histidine tag (SEQID NO:5), a sequence encoding a KEX2 protease recognition site (AAAAGA),a sequence encoding hG-CSF with its codon usage optimized for expressionin E. coli, (SEQ ID NO:2) and a Xba I digestion site.

Fragment 3, consisting of a Nde I digestion site, a sequence encodingthe OmpA signal peptide (SEQ ID NO:3), a sequence encoding hG-CSF withits codon usage optimized for expression in E. coli, (SEQ ID NO:2) and aBam HI digestion site.

Fragment 4, consisting of a Bam HI digestion site, the Kozak consensussequence (Kozak, M. J Mol Biol 1987 Aug. 20; 196(4):947-50), a sequenceencoding the hG-CSF signal peptide (SEQ ID NO:7) and hG-CSF with itscodon usage optimized for expression in CHO cells (SEQ ID NO:8) and aXba I digestion site.

DNA fragment 1 and 2 were inserted into the Bam HI and Xba I digestionsites in plasmid pJSO37 (Okkels, Ann. New York Acad. Sci. 782:202-207,1996) using standard DNA techniques. This resulted in plasmidspG-CSFcerevisiae and pHISG-CSFcerevisiae.

DNA fragment 3 was inserted into the Nde I and Bam HI digestion sites inplasmid pET12a (Invitrogen) using standard DNA techniques. This resultedin plasmid pG-CSFcoli

DNA fragment 4 was inserted into the Barn HI and Xba I digestion sitesin plasmid pcDNA3.1 (+) (Invitrogen) using standard DNA techniques. Thisresulted in plasmid pG-CSFCHO.

EXAMPLE 2

Expression of hG-CSF in S. cerevisiae and E. coli

Transformation of Saccharomyces cerevisiae YNG318 (available from theAmerican Type Culture Collection, Va., USA as ATCC 208973) with eitherplasmid pG-CSFcerevisiae or pHISG-CSFcerevisiae, isolation oftransformants containing either of the two plasmids, and subsequentextracellular expression of hG-CSF without and with the HIS tag,respectively, was performed using standard techniques described in theliterature. Transformation of E. coliBL21 (DE3) (Novagen, Cat. No.69387-3) with pG-CSFcoli, isolation of transformants containing theplasmid and subsequent expression of hG-CSF in the supernatant and inthe periplasm of the cell was performed as described in the pET SystemManual (8^(th) edition) from Novagen.

Expression of hG-CSF by S. cerevisiae and E. coli was verified byWestern Blot analysis using the ImmunoPure Ultra-Sensitive ABC RabbitIgG Staining kit (Pierce) and a polyclonal 1s antibody against hG-CSF(Pepro Tech EC Ltd.). It was observed that the protein had the correctsize.

The expression levels of hG-CSF with and without the N-terminalhistidine tag in S. cerevisiae and E. coli were quantified using acommercially available G-CSF specific ELISA kit (Quantikine Human G-CSFImmunoassay, R&D Systems Cat. No. DCS50). The measured values are listedbelow.

Expression level Expression system (mg G-CSF per L) hG-CSF in S.cerevisiae 30 hG-CSF with histidine tag in S. cerevisiae 25 hG-CSF in E.coli 0.05

EXAMPLE 3

Generation of a Stable CHO-K1 G-CSF Producer

The day before transfection the CHO K1 cell line (ATCC #CC1-61) isseeded in a T-25 flask in 5 ml DMEM/F-12 medium (Gibco # 31330-038)supplemented with 10% FBS and penicillin/streptomycin. The following day(at nearly 100% confluency) the transfection is prepared: 90 μl DMEMmedium without supplements is aliquoted into a 14 ml polypropylene tube(Corning). 10 μl Fugene 6 (Roche) is added directly into the medium andincubated for 5 min at room temperature. In the meantime 5 μg plasmidpG-CSFCHO is aliquoted into another 14 ml polypropylene tube. Afterincubation the Fugene 6 mix is added directly to the DNA solution andincubated for 15 min at room temperature. After incubation the wholevolume is added drop-wise to the cell medium.

The next day the medium is exchanged with fresh medium containing 360μg/ml hygromycin (Gibco). Every day hereafter the selection medium isrenewed until the primary transfection pool has reached 100% confluency.The primary transfection pool is sub-cloned by limited dilution (300cells seeded in five 96-well plates).

EXAMPLE 4

Purification of hG-CSF and Variants Thereof from S. cerevisiae CultureSupernatants

Purification of hG-CSF was performed as follows:

Cells are removed by centrifugation. Cell depleted supernatant is thenfilter sterilized through a 0.22 μm filter. Filter sterilizedsupernatant is diluted 5 fold in 10 mM sodium acetate pH 4.5. pH isadjusted by addition of 10 ml concentrated acetic acid per 5 liters ofdiluted supernatant. The ionic strength should be below 8 mS/cm beforeapplication to the cation exchange column.

Diluted supernatant is loaded at a linear flow rate of 90 cm/h onto aSP-sepharose FF (Pharmacia) column equilibrated with 50 mM sodiumacetate, pH 4.5 until the effluent from the column reaches a stable UVand conductivity baseline. To remove any unbound material, the column iswashed using the equilibration buffer until the effluent from the columnreaches a stable level with respect to UV absorbance and conductivity.The bound G-CSF protein is eluted from the column using a lineargradient; 30 column volumes; 0-80% buffer B (50 mM NaAc, pH 4.5, 750 mMNaCl) at a flow rate of 45 cm/h. Based on SDS-polyacryl amide gelelectrophoresis, fractions containing G-CSF are pooled. Sodium chlorideis added until the ionic strength of the solution is more than 80 mS/cm.

The protein solution is applied onto a Phenyl Toyo Pearl 650S columnequilibrated with 50 mM NaAc, pH 4.5, 750 mM NaCl. Any unbound materialis washed off the column using the equilibration buffer. Elution ofG-CSF is performed by applying a step gradient of MilliQ water.Fractions containing G-CSF are pooled. By using this 2-step down streamprocessing strategy, more than 90% pure G-CSF can be obtained. Thepurified protein is then quantified using spectrophotometricmeasurements at 280 nm and/or by amino acid analysis.

Fractions containing G-CSF are pooled. Buffer exchange and concentrationis performed using VivaSpin concentrators (mwco: 5 kDa).

EXAMPLE 5

Identification and Quantification of Non-Conjugated and ConjugatedhG-CSF and Variants Thereof

SDS-Polyacryl Amide Gel Electrophoresis

The purified, concentrated G-CSF was analyzed by SDS-PAGE. A single bandhaving an apparent molecular weight of approx. 17 kDa was dominant.

Absorbance

An estimate of the G-CSF concentration is obtained by spectrophotometricmethods. By measuring the absorbance at 280 m and using a theoreticallyextinction coefficient of 0.83, the protein concentration can becalculated.

Amino Acid Analysis

A more accurate protein determination can be obtained by amino acidanalysis. Amino acid analysis performed on a purified G-CSF revealedthat the experimentally determined amino acid composition is inagreement with the expected amino acid composition based on the DNAsequence.

EXAMPLE 6

MALDI-TOF Mass Spectrometry of PEGylated wt G-CSF and G-CSF Variants

MALDI-TOF mass spectrometry was used to evaluate the number ofPEG-groups attached to PEGylated wt G-CSF and to selected PEGylatedG-CSF variants.

Wt G-CSF contains 5 primary amines that are the expected attachmentsites for SPA-PEG (the N-terminal amino-group and the ε-amino-group onK16, K23, K34 and K40). Following PEGylation of wt G-CSF with SPA-PEG5000, MALDI-TOF mass spectrometry showed the presence of species of wtG-CSF with mainly 4, 5 and 6 PEG-groups attached. In addition, wt G-CSFwith 7 PEG-groups attached was clearly seen although in minor amounts.

The G-CSF variant having the substitutions K16R, K34R, K40R, Q70K, Q90K,and Q120K also contains 5 primary amines (the N-terminal amino-group andthe ε-amino-group on K23, K70, K90 and K120). Following PEGylation ofthis G-CSF variant with SPA-PEG5000, MALDI-TOF mass spectrometry showedthe presence of species of the G-CSF variant with mainly 4, 5 and 6PEG-groups attached. In addition, the G-CSF variant with 7 PEG-groupsattached was clearly seen although in minor amounts.

The G-CSF variant having the substitutions K16R, K34R, and K40R contains2 primary amines (the N-terminal amino-group and the ε-amino-group onK23). Following PEGylation of this G-CSF variant with SPA-PEG 12000,MALDI-TOF mass spectrometry showed the presence of species of the G-CSFvariant with mainly 2 and 3 PEG-groups attached. In addition, the G-CSFvariant with 4 PEG-groups attached was clearly seen although in minoramounts.

These observations clearly show that in addition to amino acid residuescontaining amine groups, other amino acid residues are sometimesPEGylated under the PEGylation conditions used. It also shows that it isof some importance for the PEGylation where amine groups are introduced.This has also been observed using SDS-PAGE analysis of wt G-CSF andG-CSF variants.

As described in Example 12, it has been shown that histidine 170 isfully PEGylated when the SPA-PEG chemistry is used. Furthermore, K23 andS159 are partly PEGylated. This explains the presence of 1-2 extraPEGylation sites besides the primary amines in hG-CSF and the variantsthat have been made.

EXAMPLE 7

Peptide Mapping of PEGylated and Non-PEGylated G-CSF Variants

In order to map the additional attachment sites for SPA-PEG on G-CSF andG-CSF variants the following strategy was used.

A G-CSF variant with a low number of amine groups was chosen in order toreduce the number of expected PEGylation sites to a minimum. The G-CSFvariant chosen has the substitutions K16R, K34R, K40R and H170Q. Apartfrom the e-amino-group on K23 that previous data had shown not to bePEGylated to any large extent, this variant only contains one primaryamine at the N-terminal. Thus, the background PEGylation on amine groupsis significantly reduced in this G-CSF variant. The G-CSF variant wasPEGylated using SPA-PEG 5000. Following PEGylation, the G-CSF variantwas denatured, the disulphide bonds reduced, the resulting thiol groupsalkylated, and the alkylated and PEGylated protein degraded with aglutamic acid-specific protease. Finally, the resulting peptides wereseparated by reversed phase HPLC.

Parallel with this, the non-PEGylated version of the G-CSF variant withthe substitutions K16R, K34R, and K40R was treated identically in orderto create a reference HPLC chromatogram.

Comparison of the HPLC chromatograms of the degradation of the PEGylatedG-CSF variant and the non-PEGylated G-CSF variant should then revealwhich peptides disappear upon PEGylation. Identification of thesepeptides by N-terminal amino acid sequencing of the peptide from thenon-PEGylated G-CSF variant then indirectly points to the positions thatare PEGylated.

In principle, it would have been preferable to use the non-PEGylatedversion of the G-CSF variant having all the substitutions K16R, K34R,K40R and H170Q, but for all practical purposes this does not matter.

More specifically, approximately 1 mg of the PEGylated G-CSF varianthaving the substitutions K16R, K34R, K40R and H170Q and approximately500 μg of the non-PEGylated G-CSF variant having the substitutions K16R,K34R, and K40R were dried in a SpeedVac concentrator. The two sampleswere each dissolved in 400 μl 6 M guanidinium, 0.3 M Tris-HCl, pH 8.3and denatured overnight at 37° C. Following denaturation, the disulfidebonds in the proteins were reduced by addition of 50 μl 0.1 M DTT in 6 Mguanidinium, 0.3 M Tris-HCl, pH 8.3. After 2 h of incubation at ambienttemperature the thiol groups present were alkylated by addition of 50 μl0.6 M iodoacetamid in 6 M guanidinium, 0.3 M Tris-HCl, pH 8.3.Alkylation took place for 30 min at ambient temperature before thereduced and alkylated proteins were buffer changed into 50 mM NH₄HCO₃using NAP5 columns. The volumes of the samples were reduced toapproximately 200 μl in a SpeedVac concentrator before addition of 20 μgand 10 μg glutamic acid-specific protease, respectively. Thedegradations with glutamic acid-specific protease were carried out for16 h at 37° C. The resulting peptides were separated by reversed phaseHPLC employing a Phenomenex Jupiter C₁₈ column (0.2*5 cm) eluted with alinear gradient of acetonitrile in 0.1% aqueous TFA. The collectedfractions were analyzed by MALDI-TOF mass spectrometry and subsequentlyselected peptides were subjected to N-terminal amino acid sequenceanalysis.

Comparison of the HPLC chromatograms of the degradations of thePEGylated G-CSF variant and the non-PEGylated G-CSF variant revealedthat only two fractions disappear upon PEGylation. N-terminal amino acidsequence analysis of the two fractions from the non-PEGylated G-CSFvariant showed that the peptides both were derived from the N-terminalof G-CSF. One peptide consisted of amino acid residues 1-11 generated byan unexpected cleavage following Gln 11. The other peptide consisted ofamino acid residues 1-19 generated by an expected cleavage followingGlu19.

It was expected that the N-terminal peptide of G-CSF would be identifiedusing this approach, as the N-terminal amino group is easily PEGylated.However, none of the additional attachment sites for SPA-PEG 5000 wereidentified using this approach.

An alternative to the indirect identification of PEG 5000 attachmentsites is direct identification of the attachment sites in PEGylatedpeptides. However, the fractions containing the PEGylated peptides inthe HPLC separation of the degraded PEGylated G-CSF variant are poorlyseparated from each other and from several fractions containingnon-PEGylated peptides. Thus, N-terminal amino acid sequence analysis ofthese fractions did not result in any useful data except for anindication that K23 could be partially PEGylated.

To overcome these problems, two pools of PEGylated peptides were madefrom the fractions from the first HPLC separation. These two pools weredried in a SpeedVac concentrator, dissolved in 200 μl freshly prepared50 mM NH₄HCO₃ and further degraded with 1 μg of chymotrypsin. Theresulting peptides were separated by reversed phase HPLC employing aPhenomenex Jupiter C₁₈ column (0.2*5 cm) eluted with a linear gradientof acetonitrile in 0.1% aqueous TFA. The collected fractions wereanalyzed by MALDI-TOF mass spectrometry and subsequently selectedpeptides were subjected to N-terminal amino acid sequence analysis.

From the N-terminal amino acid sequence determinations it could bedetermined that K23 as well as S159 are partially PEGylated. It was notpossible to determine the exact degree of PEGylation at these twopositions, but the PEGylation is only partial as peptides where K23 andS159 are unmodified were identified and sequenced from the initial HPLCseparation.

EXAMPLE 8

Glycosylation of wt G-CSF and G-CSF Variants

A consistent observation when analyzing purified wt G-CSF and G-CSFvariants by MALDI-TOF mass spectrometry is the presence of an additionalcomponent with a mass approximately 324 Da larger than the mass of theG-CSF molecule analyzed. As the component with the lowest massinvariantly has the mass of the G-CSF molecule and because the G-CSFmolecules have the correct N-terminal amino acid sequence, it wasconcluded that the additional component is a modified G-CSF moleculecarrying two hexose residues. In many cases the unmodified G-CSFmolecule gives rise to the most intense signal but in some cases theintensity of the signal for the modified G-CSF molecule is the mostintense.

During the analysis of the peptides generated with the aim ofidentifying the additional PEGylation sites, two peptides of interestfor identifying the site of glycosylation were identified in each of thedegradations.

In both HPLC separations, the two peptides elute next to each other andMALDI-TOF mass spectrometry shows a mass difference between the twopeptides of approximately 324 Da. The mass spectrometry data indicatesthat the peptide covers amino acid residues 124-162. N-terminal aminoacid sequence analysis of all four peptides showed that this assignmentis correct and that Thr133 is the only site of modification. In thepeptides with the mass of the unmodified peptide, Thr133 is clearly seenin the sequence, while no amino acid residue can be assigned at position133 in the peptides with an additional mass of 324 Da. As all otheramino acid residues could be assigned in the sequence, it was concludedthat Thr133 is the only site of modification. This glycosylation sitewas previously reported to be used in recombinant G-CSF expressed in CHOcells, although the glycan is different from the one attached by yeast.

The non-glycosylated wt G-CSF has been separated from the glycosylatedwt G-CSF, employing reversed phase HPLC using a Vydac C₁₈ column (0.21*5cm) isocratically eluted with 51% acetonitrile in 0.1% TFA, as afraction shown by MALDI-TOF mass spectrometry only to contain thenon-glycosylated form of wt G-CSF.

EXAMPLE 9

Separation of G-CSF Molecules with Different Numbers of PEG MoleculesCovalently Attached

Separation of G-CSF molecules covalently attached to 4, 5 or 6PEG-groups was obtained as follows. PEGylated protein in 20 mM sodiumcitrate, pH 2.5 was applied to an SP-sepharose FF column equilibratedwith 20 mM sodium citrate pH 2.5. Any unbound material was washed offthe column. Elution was performed using a pH gradient. PEGylated G-CSFbegan to elute from the column at approx. pH 3.8 and continued to elutein fractions covering a pH span from 3.8 to 4.5.

The fractions were subjected to SDS-PAGE and mass spectrometricanalysis. These analyses indicate that G-CSF having the highest degreeof PEGylation is located in the “low pH fractions”. PEGylated G-CSFhaving a lower degree of PEGylation is eluted in the “high pHfractions”.

Amino acid analysis performed on PEGylated G-CSF showed good consistencybetween the theoretically and the experimentally determined extinctioncoefficient.

EXAMPLE 10

Construction of hG-CSF Variants

Specific substitutions of existing amino acids in hG-CSF to other aminoacid residues, e.g. the specific substitutions discussed above in thegeneral description, were introduced using standard DNA techniques knownin the art. The new G-CSF variants were made using plasmidpG-CSFcerevisiae containing the gene, encoding hG-CSF without the HIStag, as DNA template in the PCR reactions. The variants were expressedin S. cerevisiae and purified as described in Example 4. Some of theconstructed G-CSF variants are listed below (see Examples 12 and 13).

EXAMPLE 11

Covalent Attachment of SPA-PEG to hG-CSF or Variants Thereof.

Human G-CSF and variants thereof were covalently linked to SPA-PEG 5000,SPA-PEG 12000 and SPA-PEG 20000 (Shearwater) as described above(“PEGylation of hG-CSF and variants thereof in solution”). The in vitroactivities of the conjugates are listed in Example 13.

EXAMPLE 12

Identification of SPA-PEG Attachment Sites in G-CSF by Site-DirectedMutagenesis Followed by PEGylation of the Purified Variants

SPA-PEG may be attached to other amino acid residues than lysine inG-CSF. In order to determine whether SPA-PEG was attached to histidines,serines, threonines and arginines, variants were made in which theseamino acids were substituted to lysine, alanine or glutamine. Thevariants were expressed in S. cerevisiae, purified and PEGylatedfollowed by analysis of the number of attached SPA-PEG molecules onSDS-PAGE. This analysis was performed by visual inspection of theSDS-PAGE gels, all of which contained three major bands. The degree ofPEGylation was estimated to the nearest 5% for each band based on therelative size of the bands. A reduction in the number of attachedSPA-PEG molecules after substitution of a given amino acid withglutamine or alanine strongly indicates that this amino acid isPEGylated by SPA-PEG, and this observation is further supported by anunchanged degree of PEGylation after substitution of the amino acid tolysine. The analyzed variants are listed below.

G-CSF variant No. of attached PEG groups hG-CSF 10% 4 PEG, 75% 5 PEG,15% 6 PEG K23R 10% 4 PEG, 85% 5 PEG, 5% 6 PEG H43Q 10% 4 PEG, 75% 5 PEG,15% 6 PEG H43K 10% 5 PEG, 75% 6 PEG, 15% 7 PEG H52Q 10% 4 PEG, 75% 5PEG, 15% 6 PEG H52K 10% 5 PEG, 75% 6 PEG, 15% 7 PEG H156Q 10% 4 PEG, 75%5 PEG, 15% 6 PEG H156K 10% 5 PEG, 75% 6 PEG, 15% 7 PEG H170Q 10% 3 PEG,75% 4 PEG, 15% 5 PEG H170K 10% 4 PEG, 75% 5 PEG, 15% 6 PEG K16/34R 10% 2PEG, 75% 3 PEG, 15% 4 PEG K16/34R R22K 10% 3 PEG, 75% 4 PEG, 15% 5 PEGK16/34R R22Q 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34R S142A 10% 2 PEG,75% 3 PEG, 15% 4 PEG K16/34/40R 10% 1 PEG, 75% 2 PEG, 15% 3 PEGK16/34/40R S53K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S53A 10% 2PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S62K 10% 2 PEG, 75% 3 PEG, 15% 4PEG K16/34/40R S66K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S80K 10%2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R T105K 10% 2 PEG, 75% 3 PEG, 15% 4PEG K16/34/40R T133K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R S142K10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40R R147K 10% 2 PEG, 75% 3 PEG,15% 4 PEG K16/34/40R S155K 10% 2 PEG, 75% 3 PEG, 15% 4 PEG K16/34/40RS159K 10% 2 PEG, 85% 3 PEG, 5% 4 PEG K16/34/40R S170K 10% 1 PEG, 75% 2PEG, 15% 3 PEG

The data show that besides the N-terminus, K16, K34 and K40, SPA-PEGalso is covalently bound to H170. Furthermore, the data show that only10% of the available K23 amino acid residues are PEGylated, and thatapproximately 10% of S159 is PEGylated.

EXAMPLE 13

In Vitro Biological Activity of Non-Conjugated and Conjugated hG-CSF andVariants Thereof.

The in vitro biological activities of conjugated and non-conjugatedhG-CSF and variants thereof were measured as described above in “Primaryassay 2—in vitro hG-CSF activity assay”. The in vitro bioactivities,represented by the measured EC50 values for each variant with andwithout conjugation of SPA-PEG 5000 to the available PEGylation sites,are listed below. The values have been normalized with respect to theEC50 value of non-conjugated hG-CSF (Neupogen®), i.e. the values in thetable indicate % activity relative to the activity of non-conjugatedhG-CSF. This value was measured simultaneously with the variants eachtime under identical assay conditions. The EC50 value of hG-CSF in thedescribed assay is 30 pM.

EC50 (% of hG-CSF) EC50 (% of hG-CSF) conjugated to G-CSF variantnon-conjugated SPA-PEG 5000 G-CSF with N-terminal Histidine tag 10 Notdetermined G-CSF without N-terminal Histidine tag 100 0.1 16R 100 1 16Q80 1 23Q 80 0.1 23R 100 0.1 34R 100 1 34A 80 1 34Q 70 1 40R 50 1 K16/23R100 1 K16/23Q 80 1 K34/40R 50 5 K16/34R 100 10 K16/40R 50 5 K16/23/34R50 10 K16/23/40R 50 5 K16/34/40R 35 30 K16/23/34/40R 20 15 K16/34/40RL3K 50 25 K16/34/40R E45K Expressed at low levels Not determinedK16/34/40R E46K 10 1 K16/34/40R S53K 5 0.5 K16/34/40R S62K 10 0.5K16/34/40R S66K 20 2 K16/34/40R Q67K 10 0.2 K16/34/40R Q70K 30 20K16/34/40R S76 50 20 K16/34/40R Q77 1 0 K16/34/40R S80K 10 0.2K16/34/40RQ90K 30 20 K16/34/40R E98K Expressed at low levels Notdetermined K16/34/40R D104K 10 0.9 K16/34/40R T105K 30 10 K16/34/40RQ120K 30 20 K16/34/40R Q131K Expressed at low levels Not determinedK16/34/40R T133K 30 10 K16/34/40R Q134K 10 0.2 K16/34/40R S142K 20 7K16/34/40R R147K 20 1 K16/34/40R S155K 20 1 K16/34/40R Q158 20 5K16/34/40R S159K 20 3 K16/34/40R Q70K Q90K Not determined 20 K16/34/40RQ70K Q120K 25 25 K16/34/40R Q90K T105K 40 10 K16/34/40R Q90K Q120K 25 15K16/34/40R Q90K S159K 45 Not determined K16/34/40R T105K Q120K 20 8K16/34/40R T105K S159K 40 20 K16/34/40R Q120K T133K 20 8 K16/34/40RQ120K S142K 10 2 K16/34/40R Q70K Q90K T105K 10 4 K16/34/40R Q70K Q90KQ120K 20 12 K16/34/40R Q70K Q90K T133K 15 5 K16/34/40R Q70K T105K Q120K10 2 K16/34/40R Q70K Q120K T133K 15 2 K16/34/40R Q70K Q120K S142K 10 1K16/34/40R Q90K T105K Q120K 10 2 K16/34/40R Q90KT105K T133K 10 2K16/34/40R Q90K T105K S159K 55 5 K16/34/40R Q90K Q120K T133K 15 2K16/34/40R Q90K Q120K S142K 10 1 K16/34/40R T105K Q120K T133K 10 1K16/34/40R Q120K T133K S142K 10 1

The data show that substitution of K23 to arginine does not increase theactivity of the conjugated protein. This is due to the fact that only10% of K23 is PEGylated, whereby the conjugated K23R variant hasessentially the same number of PEG groups attached to it and has thesame location of the PEGylation sites as hG-CSF. Removal of theremaining lysines at position K16, K34 and K40 resulted in a G-CSFvariant with significant activity after PEGylation. Conjugation ofSPA-PEG 5000 to this variant does not decrease the activitysignificantly as compared to the non-conjugated variant. Thus,PEGylation of the N-terminus and H170 with SPA-PEG 5000 (see Example 12)does not decrease the activity of hG-CSF. It was decided to use hG-CSFK16R K34R K40R as the backbone for insertion of new PEGylation sites. 24new PEGylation sites between residues L3 and H 159 were introduced inthis backbone. These residues are distributed over the parts of hG-CSFthat do not interact with the G-CSF receptor. Introduction of newPEGylation sites at positions L3, Q70, S76, Q90, T105, Q120, T133 andS142 resulted in hG-CSF variants that retained a significant amount ofactivity after PEGylation by SPA-PEG 5000. Thus, some of these newPEGylation sites were combined in hG-CSF variants that had 2 or 3 newPEGylation sites.

Furthermore, SPA-PEG 12000 and SPA-PEG 20000 were attached to a group aselected hG-CSF variants. The in vitro activities are listed below (% ofNeupogen®).

EC50 EC50 (% of hG-CSF) (% of hG-CSF) conjugated to conjugated to G-CSFvariant SPA-PEG 12000 SPA-PEG 20000 K16/34/40R 10 1 K16/34/40R Q90K Notdetermined 7 K16/34/40R Q70K Q90K 8 Not determined K16/34/40R Q90K T105K1 <1 K16/34/40R T105K S159K 6 5 K16/34/40R Q90K T105K S159K 1 <1

EXAMPLE 14

In Vivo Half-Life of Non-Conjugated and Conjugated hG-CSF and VariantsThereof.

The in vivo half-lives of non-conjugated hG-CSF (Neupogen®), SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K and SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q90K T105K S159K were measured asdescribed above (“Measurement of the in vivo half-life of conjugated andnon-conjugated rhG-CSF and variants thereof”). The results are shown inFIGS. 1 and 2. The in vivo half-life of Neupogen® was determined to be2.01 hours and 1.40 hours, respectively. In an earlier, similarexperiment (U.S. Pat. No. 5,824,778), the in vivo half-life of hG-CSFwas determined to be 1.79 hours. The results of the experimentsdescribed herein can therefore be directly compared to that experiment.The in vivo half-lives of SPA-PEG 5000 conjugated hG-CSF K16R K34R K40RQ70K Q90K Q120K and SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q90KT105K S159K were determined to be 12.15 hours and 16.10 hours,respectively. Thus, introducing new PEGylation sites in hG-CSF andconjugating SPA-PEG 5000 to them has resulted in a significant increasein the in vivo half-life.

In the earlier experiment described above (U.S. Pat. No. 5,824,778), thein vivo half-life of hG-CSF conjugated to a larger N-terminally attachedPEG molecule (10 kDa) was determined to be 7.05 hours. Thus, SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K and SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q90K T105K S159K have significantlylonger half-lives than both Neupogen® and hG-CSF with a 10 kDaN-terminally conjugated PEG molecule. SPA-PEG 5000 conjugated hG-CSFK16R K34R K40R Q70K Q90K Q120K and SPA-PEG 5000 conjugated hG-CSF K16RK34R K40R Q90K T105K S159K both have three removed endogenous PEGylationsites and three new introduced PEGylation sites and thus are identicalin size. The only difference between the two compounds is the in vitroactivity, which is 12% and 5%, respectively, of that of Neupogen®. Thisdifference results in a longer in vivo half-life of SPA-PEG 5000conjugated K16R K34R K40R Q90K T105K S159K compared to SPA-PEG 5000conjugated K16R K34R K40R Q70K Q90K Q120K. Since the in vitro activitiescorrelate with the receptor binding affinities of the compounds, it canbe concluded that the receptor-mediated clearance of SPA-PEG 5000conjugated K16R K34R K40R Q90K T105K S159K is slower than that ofSPA-PEG 5000 conjugated K16R K34R K40R Q70K Q90K Q120K. Thus, acombination of increasing the size and reducing the in vitro activity ofG-CSF results in G-CSF compounds with significantly longer in vivohalf-lives than previously described compounds.

EXAMPLE 15

In Vivo Biological Activity in Health Rats of Non-Conjugated andConjugated hG-CSF and Variants Thereof.

The in vivo biological activities of non-conjugated hG-CSF (Neupogen®),SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q120K, SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K, SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q120K T133K and SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q90K Q120K T133K were measured asdescribed above (“Measurement of the in vivo biological activity inhealthy rats of conjugated and non-conjugated hG-CSF and variantsthereof”). The results are shown in FIGS. 3 and 4.

No activity of Neupogen® could be detected at 48 hours after injectionof 100 μg per kg body weight at t=0 hours. Activity of SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q120K, SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q70K Q120K T133K and SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q90K Q120K T133K could be detected until 72 hoursafter the initial injection, while SPA-PEG 5000 conjugated hG-CSF K16RK34R K40R Q70K Q90K Q120K remained active in vivo until 96 hours afterthe initial injection. Thus, it was shown that all of these conjugatedvariants had a longer in vivo biological activity than Neupogen® andthat SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120Kremained active twice as long in vivo as Neupogen®. SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q120K T133K and SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q90K Q120K T133K, both with an in vitroactivity of 2% of Neupogen® (Example 13), did not induce the same levelof white blood cell formation during the initial 12 hours afteradministration as observed after administration of Neupogen®, SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q120K and SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K. Thus, the compoundswith an in vitro activity of 2% or less compared to that of Neupogen®were unable to stimulate full formation of white blood cells immediatelyafter administration.

Furthermore, the in vivo biological activities of Neupogen®, SPA-PEG12000 conjugated hG-CSF K16R K34R K40R and different doses of SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K were measured asdescribed above (“Measurement of the in vivo biological activity inhealthy rats of conjugated and non-conjugated hG-CSF and variantsthereof”). The results are shown in FIG. 5. As observed earlier, noactivity of Neupogen® could be detected 48 hours after the initialinjection of 100 μg per kg body weight. Administration of 5 μg per kgbody weight of SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90KQ120K resulted in a slightly longer in vivo biological activity thanNeupogen®, while administration of 25 μg per kg body weight and 100 μgper kg body weight of this compound resulted in hG-CSF activity until 72and 96 hours, respectively, after the initial injection. Thus, theduration of action of the SPA-PEG conjugated hG-CSF compounds can becontrolled by increasing or decreasing the standard dosing regimen.SPA-PEG 12000 conjugated hG-CSF K16R K34R K40R remained active in vivountil 72 hours after administration of 100 μg per kg body weight. Asdescribed in Example 6, SPA-PEG 12000 conjugated hG-CSF K16R K34R K40Rhas 2 or 3 SPA-PEG 12000 groups attached while SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q70K Q90K Q120K has 5 or 6 SPA-PEG 5000 groupsattached. Thus, the molecular weights of the two compounds are 42-54 kDaand 43-48 kDa, respectively. The in vitro activities of the twocompounds are 30% and 12%, respectively, of that of Neupogen®. Thelonger in vivo biological activity of SPA-PEG 5000 conjugated hG-CSFK16R K34R K40R Q70K Q90K Q120K as compared to SPA-PEG 12000 conjugatedhG-CSF K16R K34R K40R with essentially the same molecular weightsuggests that when the size of the G-CSF compounds is increased above acertain molecular weight through PEGylation, the duration of action canonly be increased further by reducing the specific activity of the G-CSFcompounds and thus, the receptor-mediated clearance (see Example 14).

Furthermore, the in vivo biological activities of Neupogen®, SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K Q120K, SPA-PEG 5000conjugated hG-CSF K16R 34R 40R Q90K T105K S159K and SPA-PEG 20000conjugated hG-CSF K16R 34R 40R T105K S159K were measured as describedabove (“Measurement of the in vivo biological activity in healthy ratsof conjugated and non-conjugated hG-CSF and variants thereof”). Theresults are shown in FIG. 6.

As observed earlier, the conjugated hG-CSF variants had a significantlonger duration of action than Neupogen®. Administration of each ofthese three conjugated hG-CSF variants resulted in formation of whiteblood cells at the same rate and to the same level as observed afteradministration of Neupogen® during the initial 12 hours afteradministration. The in vitro activities of SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q70K Q90K Q120K, SPA-PEG 5000 conjugated hG-CSFK16R 34R 40R Q90K T105K S159K and SPA-PEG 20000 conjugated hG-CSF K16R34R 40R T105K S159K are 12%, 5% and 5%, respectively, of that ofNeupogen®, and thus, a hG-CSF compound with 5% of Neupogen® activity invitro is able to induce full white blood cell formation afteradministration.

The apparent size on SDS-PAGE of Neupogen®, SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q70K Q90K Q120K, SPA-PEG 5000 conjugated hG-CSFK16R 34R 40R Q90K T105K S159K and SPA-PEG 20000 conjugated hG-CSF K16R34R 40R T105K S159K is 18 kDa, 60 kDa, 60 kDa and >100 kDa,respectively. SPA-PEG 5000 conjugated hG-CSF K16R 34R 40R Q90K T105KS159K and SPA-PEG 20000 conjugated hG-CSF K16R 34R 40R T105K S159K havealmost identical durations of action in vivo, showing that the durationof action is not increased by increasing the molecular size of theconjugated hG-CSF compounds above an apparent size of about 60 kDa.Instead, when the apparent size of the conjugated hG-CSF compounds isabove about 60 kDa, the duration of action may be increased be reducingthe in vitro activity and hence, the receptor binding affinity of thecompound. An additional example of this (see above) can be observed bycomparing the in vivo duration of action of SPA-PEG 5000 conjugatedhG-CSF K16R 34R 40R Q70K Q90K Q120K and SPA-PEG 5000 conjugated hG-CSFK16R34R 40R Q90K T105K S159K. The two compounds both have an apparentsize of 60 kDa, while the in vitro activities are 12% and 5%,respectively. This difference is reflected directly in the in vivoduration of action of the two compounds, which is 96 hours and 144hours, respectively.

EXAMPLE 16

In Vivo Biological Activity in Rats with Chemotherapy-InducedNeutropenia of Non-Conjugated and Conjugated hG-CSF and VariantsThereof.

The in vivo biological activities in rats with chemotherapy-inducedneutropenia of non-conjugated hG-CSF (Neupogen®), SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K and SPA-PEG 20000conjugated hG-CSF K16R K34R K40R Q90K were measured as described above(“Measurement of the in vivo biological activity in rats withchemotherapy-induced neutropenia of conjugated and non-conjugated hG-CSFand variants thereof”) using 50 mg per kg body weight of CPA and asingle dose (100 μg per kg body weight) of G-CSF. The results are shownin FIG. 7. The three compounds induced an initial formation of whiteblood cells with identical rates. Thus, an in vitro activity of 4% ofthat of Neupogen® is sufficient for a conjugated hG-CSF compound to givefull stimulation of white blood cell formation in vivo immediately afteradministration. After 36 hours the number of white blood cells (WBC) inthe Neupogen®-treated rats dropped to the level that was observed in theuntreated group (<3×10⁹ cells per litre). At this point the rats wereneutropenic. The level of WBC in both groups reached normal levels(9×10⁹ cells per litre) after 144 hours.

The level of WBC in the two groups treated with SPA-PEG 5000 conjugatedhG-CSF K16R K34R K40R Q70K Q90K T105K and SPA-PEG 20000 conjugatedhG-CSF K16R K34R K40R Q90K dropped to a minimum of 4×10⁹ cells per litreafter 48 hours and then immediately started to increase. The WBC levelsin both groups were back to normal after 96 hours. Thus, the twoconjugated hG-CSF compounds were able to both relieve the degree ofneutropenia and to reduce the time until the WBC levels were back tonormal (the duration of neutropenia) significantly from 112 hours in theNeupogen®-treated group to 48 hours in the groups treated with eitherSPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K andSPA-PEG 20000 conjugated hG-CSF K16R K34R K40R Q90K.

SPA-PEG 5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K moreefficiently shortened the duration of neutropenia as compared to SPA-PEG20000 conjugated hG-CSF K16R K34R K40R Q90K. Since the apparent size ofboth molecules is above 60 kDa (60 kDa and 80 kDa, respectively) thiscannot be explained by a lower renal clearance of SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K than SPA-PEG 20000conjugated hG-CSF K16R K34R K40R Q90K. The in vitro activity of SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K and SPA-PEG 20000conjugated hG-CSF K16R K34R K40R Q90K are 4% and 7% of Neupogen®,respectively. This means that the receptor binding affinity and thus,the receptor-mediated clearance, of SPA-PEG 5000 conjugated hG-CSF K16RK34R K40R Q70K Q90K T105K is lower than for SPA-PEG 20000 conjugatedhG-CSF K16R K34R K40R Q90K in the initial 48 hours after administrationwhere the white blood cell levels are increased. Hence, when the ratsbecome neutropenic after 48 hours, the in vivo concentration of SPA-PEG5000 conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K is higher thanSPA-PEG 20000 conjugated hG-CSF K16R K34R K40R Q90K. Since a relativelylow in vitro G-CSF activity of 4-5% of that of Neupogen® is sufficientto obtain full activation of the G-CSF receptors on the neutrophilprogenitor cells (see above), this higher G-CSF concentration after 48hours explains the faster increase in WBC levels in the SPA-PEG 5000conjugated hG-CSF K16R K34R K40R Q70K Q90K T105K-treated group. Thus, inrats with chemotherapy-induced neutropenia, a conjugated G-CSF compoundof the invention with an apparent size of at least about 60 kDa and anin vitro activity of 4% of that of Neupogen® is superior to similar sizecompounds with a higher in vitro activity.

EXAMPLE 17

Purification of G-CSF from S. cerevisiae Culture Supernatants

This example provides an alternative purification procedure to that ofExample 4 for purification of hG-CSF and G-CSF variants.

Cells are removed by centrifugation, 5000 rpm, 10 min, 4° C., and theclarified supernatant is filtered through a 0.22 μm filter. Theclarified and filtered supernatant is concentrated and diafiltered into50 mM sodium acetate, pH 4.5, by Tangential Flow Filtration using 10 kDamembranes.

The resulting ultra filtrate is applied onto an SP-sepharose column (200ml packed bed) equilibrated with at least 5 column volumes of 50 mMsodium acetate. Samples are loaded at a flow rate of approx. 20 ml/min.The column is washed using the equilibration buffer until a stableeffluent is obtained as determined by absorbance at 280 nm. Using astepwise buffer gradient (e.g. 10%, 20%, 30% and 35% buffer), G-CSF iseluted at 35% buffer at ambient flow rate, where the buffer is 750 mMNaCl in 50 mM sodium acetate.

This one-step method yields >95% pure G-CSF (as determined by SDS-PAGE).

EXAMPLE 18

Separation of Multi-PEGylated Species of G-CSF

Example 9 above describes a method for separation of G-CSF moleculeswith different numbers of PEG groups attached. This example provides analternative procedure for separation of such multi-PEGylated G-CSFspecies in order to obtain a G-CSF product with a desired degree ofuniformity in terms of the number of attached PEG groups.

A mixture of PEGylated G-CSF, covalently linked to e.g. SPA-PEG 5000(Shearwater) as described above (“PEGylation of hG-CSF and variantsthereof in solution”), is diluted with 20 mM citrate buffer, pH 2.5. Theconductivity should be <3.5 mS/cm. The pH is adjusted to 2.5 asnecessary using dilute HCl. The following buffers are used for theseparation:

-   Buffer A: 20 mM sodium citrate, pH 2.5 (equilibration and washing    buffer).-   Buffer B: 20 mM sodium citrate, pH 2.5; 750 mM sodium chloride    (elution buffer)

The sample to be separated is loaded onto an equilibrated SP-sepharoseHP column (7 ml) at a flow rate of 2 ml/min. The column is washed withBuffer A until a stable baseline is obtained as monitored by A₂₈₀.

Multi-PEGylated species are separated by applying a linear gradient of0-50% Buffer B for 180 minutes at a flow rate of 4 ml/min and collecting2 ml fractions. The collected fraction are analyzed by SDS-PAGE, andfractions having a desired number of attached PEG groups are pooled.This allows purification of a PEGylated G-CSF mixture comprising speciesinitially having, e.g., 3-6 attached PEG groups to result in a producthaving e.g. only 4 or 5 PEG groups attached, or a product having only asingle number of attached PEG groups.

EXAMPLE 19

Peptide Mapping

Using a similar procedure to that described above in Example 7, butbased on degradation with trypsin, the PEGylation pattern of a G-CSFconjugate of the invention was determined by peptide mapping. In thiscase, the polypeptide was produced in CHO cells (see Example 3) and hadthe substitutions K16R, K34R, K40R, T105K and S159K relative to thesequence of native human G-CSF. It was PEGylated with 5 kDa SPA-PEG asdescribed above, resulting in modified proteins carrying predominantly3, 4 or 5 PEG moieties, and to a small extent 6 PEG moieties. Five ofthe six possible PEG attachment sites are known, these being theN-terminal amino group, Lys23, Lys105, Lys159 and His170.

This peptide mapping analysis showed that the conjugated protein wasessentially fully PEGylated at the N-terminal and at Lys105 and Lys159,while Lys23 was partially PEG-ylated. Although His 170 has been shown tobe partially PEGylated in previous experiments, this was surprisinglynot found in this experiment. One possible explanation for thisobservation is that the bond between the PEG and the His170 residue maybe unstable during the sample preparation carried out prior to thepeptide mapping. A possible unstable PEGylation such as may be the casehere may be avoided by substituting the histidine residue with anotherresidue, in particular a lysine residue if a more stable PEGylation isdesired, or a glutamine or arginine residue if PEGylation is to beavoided.

EXAMPLE 20

In Vivo Biological Activity in Rats with Chemotherapy-InducedNeutropenia

The in vivo biological activity of two PEGylated G-CSF variants of theinvention was tested in rats with chemotherapy-induced neutropenia. Thevariants had, relative to SEQ ID NO:1, the amino acid substitutionsK16R, K34R, K40R, T105K and S159K (referred to below as “105/159”) andK16R, K34R, K40R, Q90K, T105K and S159K (referred to as “90/105/159”),respectively. Both variants were produced in yeast (S. cerevisiae) andwere conjugated with SPA-PEG-5000 as described above. The in vivobiological activity of a single dose of the two variants was testedagainst the activity of daily doses of non-conjugated hG-CSF (Neupogen®)and a control (vehicle).

24 hours before administration of the G-CSF samples, the rats were given50 mg per kg body weight of CPA. The PEGylated variants of the inventionwere administered as a single dose of 100 μg per kg body weight at time0, while Neupogen® was administered in daily doses of 30 μg per kg bodyweight for 5 days (from 0 hours to 96 hours).

The in vivo biological activity was measured as described above(“Measurement of the in vivo biological activity in rats withchemotherapy-induced neutropenia of conjugated and non-conjugated hG-CSFand variants thereof”). The results are shown in FIG. 8 (white bloodcell count, WBC) and in FIG. 9 (absolute neutrophil count, ANC).

As seen in FIG. 8, administration of 105/159, 90/105/159 and Neupogen®all resulted in an initial increase in white blood cell levels in thefirst 12 hours, after which the white blood cell levels fell as a resultof the chemotherapy, reaching a minimum after about 48 hours. After 48hours, the numbers of white blood cells increased for all threetreatment groups, although the rate of increase was clearly greater forthe group treated with the two PEGylated variants of the invention thanfor the group treated with Neupogen®. Treatment with the PEGylatedvariants 105/159 and 90/105/159 resulted in a normal level of whiteblood cells (over 10×10⁹/l) after 96 hours, while the Neupogen® treatedgroup still had a white blood cell level under 10×10⁹/l after 120 hours.Since the last of the five daily Neupogen® treatments was given at 96hours, the white blood cell level in this group fell again after 120hours. In contrast, the white blood cell level in the two groups treatedwith a single dose of the PEGylated variants of the invention wasrelatively stable at just over 10×10⁹/l from 96 hours and for theduration of the experiment until 216 hours.

A similar pattern for the numbers of neutrophils is seen in FIG. 9,which shows that the neutrophil level for the group treated with thePEGylated variant 105/159 increased significantly faster than for thegroup treated with Neupogen® (ANC was not determined for the 90/105/159group).

EXAMPLE 21

In Vivo Biological Activity in Rats with Chemotherapy-InducedNeutropenia

The in vivo biological activities of non-conjugated hG-CSF (Neupogen®)and hG-CSF with a single N-terminally linked 20 kDa PEG group(Neulasta®) were compared to two PEGylated G-CSF variants of theinvention in rats with chemotherapy-induced neutropenia. These twovariants, which were produced in yeast (S. cerevisiae) and CHO cells,respectively, had the same amino acid substitutions relative to thesequence of hG-CSF, namely K16R, K34R, K40R, T105K and S159K, and wereconjugated to SPA-PEG 5000. The PEGylated variants of the invention,which initially consisted of multi-PEGylated species having 3-6 PEGmoieties attached, were separated to give a more uniform product havingonly 4-5 PEG moieties attached. These variants are referred to below as“G20” (produced in yeast) and “G21” (produced in CHO cells).

The G-CSF samples were administered 24 hours after administration of CPA(90 mg per kg body weight). The PEGylated variants, i.e. Neulasta™, G20and G21, were administered as a single dose of 100 μg per kg bodyweight, while Neupogen® was administered in daily doses of 10 μg per kgbody weight for seven days.

The in vivo biological activity was measured as described above(“Measurement of the in vivo biological activity in rats withchemotherapy-induced neutropenia of conjugated and non-conjugated hG-CSFand variants thereof”). The results are shown in FIG. 10 (white bloodcell count, WBC) and FIG. 11 (absolute neutrophil count, ANC).

FIGS. 10 and 11 show that all of the G-CSF compounds induced an initialformation of white blood cells and neutrophils at approximatelyidentical rates during the first 12 hours, after which the levels ofwhite blood cells and neutrophils fell as a result of the chemotherapy.After 96 hours, the levels of white blood cells and neutrophilsincreased once again in all cases, but the rate of increase wassignificantly higher for rats treated with G20 or G21 than for ratstreated with either Neupogen® or Neulasta®. FIG. 10 shows that the whiteblood cell levels of rats treated with G20 or G21 reached a normal levelof approximately 10⁹/l after 144 hours, while the rats treated withNeupogen® or Neulasta™ did not reach this level until after 168 hours.As shown in FIG. 11, the same pattern is seen when looking at theneutrophil count, i.e. the neutrophil count of rats treated with G20 orG21 reach a normal level approximately 24 hours before rats treated withNeupogen® or Neulasta™ reach a similar level. It may thus be concludedthat these PEGylated G-CSF variants of the invention are able to reducethe duration of chemotherapy-induced neutropenia in rats by about 24hours compared to treatment with the currently available G-CSF productsNeupogen® and Neulasta™.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. For example, all thetechniques and apparatus described above may be used in variouscombinations. All publications, patents, patent applications, and/orother documents cited in this application are incorporated herein byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent, patent application, and/or otherdocument were individually indicated to be incorporated herein byreference in its entirety for all purposes.

1. A method for preparing a G-CSF conjugate, the method comprising (a)providing a polypeptide comprising an amino acid sequence consisting ofSEQ ID NO:1 with the substitutions K16R, K34R, K40R, T105K and S159K,and (b) reacting the polypeptide with amine-reactive polyethylene glycolmoieties under conditions sufficient to produce a G-CSF conjugate havinga polyethylene glycol moiety covalently bound to each of at least twoattachment groups of the polypeptide, wherein the G-CSF conjugateexhibits G-CSF cell proliferation activity.
 2. The method of claim 1,wherein the polyethylene glycol moieties each have a molecular weight ofabout 5 kilodaltons.
 3. The method of claim 1, wherein theamine-Reactive polyethylene glycol moieties are mPEG-succinimidylpropionate (mPEG-SPA).
 4. The method of claim 3, wherein theamine-reactive polyethylene glycol moieties are mPEG-SPA
 5000. 5. Themethod of claim 1, wherein the amine-reactive polyethylene glycolmoieties are attached to the N-terminal amino group and to one or morelysine residues.
 6. The method of claim 5, wherein the one or morelysine residues are selected from Lys23, Lys105, and Lys159.
 7. Themethod of claim 1, wherein the step of providing the polypeptidecomprises: providing an isolated host cell comprising a nucleic acidthat comprises a promoter operably linked to a nucleotide sequenceencoding the polypeptide, culturing the host cell in a nutrient mediumunder conditions which permit expression of the polypeptide, andrecovering the polypeptide from the nutrient medium or from the culturedhost cells.
 8. The method of claim 7, wherein the host cell is abacterial host cell.
 9. The method of claim 7, wherein the host cell isa glycosylating host cell.
 10. The method of claim 9, wherein the hostcell is selected from an S. cerevisiae cell, a Pichia pastoris cell, aCHO cell, a BHK cell, an HEK 293 cell, and an SF9 cell.
 11. The methodof claim 8, wherein the host cell is E. coli.
 12. The method of claim10, wherein the host cell is an S. cerevisiae cell.
 13. The method ofclaim 10, wherein the host cell is a CHO cell.
 14. The method of claim7, wherein the polypeptide is recovered from the nutrient medium. 15.The method of claim 7, wherein the polypeptide is recovered from thecultured host cells.
 16. The method of claim 7, wherein the methodfurther comprises a purification step to purify the recoveredpolypeptide.
 17. The method of claim 16, wherein the purification stepcomprises a chromatography method.
 18. The method of claim 17, whereinthe chromatography method is ion exchange chromatography.
 19. The methodof claim 1, wherein the amine-reactive polyethylene glycol moietiescomprise a succinimidyl group.
 20. The method of claim 1, wherein themethod further comprises a purification step.
 21. The method of claim20, wherein the purification step comprises cation exchangechromatography.
 22. The method of claim 1, wherein the conditions are amolar excess of the amine-reactive polyethylene glycol moieties relativeto the number of polypeptide attachment sites.
 23. The method of claim1, wherein the conditions are a molar ratio of amine-reactivepolyethylene glycol moieties to polypeptide that is greater than 10:1and up to about 100:1.
 24. The method of claim 22, wherein theconditions are about a 5:1 molar excess of the amine-reactivepolyethylene glycol moieties relative to the number of polypeptideattachment sites.
 25. The method of claim 22, wherein the conditions areabout a 10:1 molar excess of the amine-reactive polyethylene glycolmoieties relative to the number of polypeptide attachment sites.
 26. Themethod of claim 22, wherein the conditions are about a 1000:1 molarexcess of the amine-reactive polyethylene glycol moieties relative tothe number of polypeptide attachment sites.
 27. The method of claim 1,wherein the G-CSF conjugate comprises 2-8 polyethylene glycol moieties.28. The method of claim 1, wherein the G-CSF conjugate comprises 3-6polyethylene glycol moieties.
 29. The method of claim 19, wherein theamine-reactive polyethylene glycol moiety is a methoxypolyethyleneglycol (mPEG) moiety.
 30. The method of claim 29, wherein theamine-reactive polyethylene glycol moiety is a linear molecule.